Triterpene saponins, methods of synthesis and uses thereof

ABSTRACT

The present invention relates to triterpene glycoside saponin-derived adjuvants, syntheses thereof, intermediates thereto, and uses thereof. QS-7 is a potent immuno-adjuvant that is significantly less toxic than QS-21, a related saponin that is currently the favored adjuvant in anticancer and antiviral vaccines. Tedious isolation and purification protocols have hindered the clinical development of QS-7. A novel semi-synthetic method is provided wherein a hydrolyzed prosapogenin mixture is used to synthesize QS-7, QS-21, and related analogs, greatly facilitating access to QS-7 and QS-21 analogs for preclinical and clinical evaluation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/613,312, filed Sep. 13, 2012, which is a divisional of andclaims priority under 35 U.S.C. § 120 to U.S. patent application Ser.No. 12/420,803, filed Apr. 8, 2009, which claims priority under 35U.S.C. § 119(e) to U.S. provisional application Ser. No. 61/043,197,filed Apr. 8, 2008, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with United States Government support undergrant GM58833 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to triterpene glycoside saponin-derivedadjuvants, syntheses thereof, and intermediates thereto. The inventionalso provides pharmaceutical compositions comprising compounds of thepresent invention and methods of using said compounds or compositions inthe treatment of infectious diseases and cancer.

BACKGROUND OF THE INVENTION

Saponins are glycosidic compounds that are produced as secondarymetabolites of steroids and triterpenes. They are widely distributedamong plant species and in some marine invertebrates. The chemicalstructure of saponins imparts a wide range of pharmacological andbiological activities, including some potent and efficaciousimmunological activity. Semi-purified saponin extracts from the bark ofthe South American Quillaja saponaria Molina tree (Quillajasaponins)exhibit remarkable immunoadjuvant activity. Because the Quillajasaponinsare found as a mixture of at least one hundred structurally relatedsaponin glycosides, their separation and isolation is often difficult ifnot prohibitive.

The most active fraction of these extracts, designated QS-21, has beenfound to include a mixture of two principal isomeric triterpeneglycoside saponins, each incorporating a quillaic acid triterpene core,flanked on either side by complex oligosaccharides and astereochemically rich glycosylated fatty acyl chain. The potency ofQS-21 and its favorable toxicity profile in dozens of recent and ongoingvaccine clinical trials (melanoma, breast cancer, small cell lungcancer, prostate cancer, HIV-1, malaria) have established it as apromising new adjuvant for immune response potentiation anddose-sparing. However, the tolerated dose of QS-21 does not exceed 100μg, above which significant local and systemic side effects arise.

Access to other potent Quillajasaponins has been hindered bydifficulties in obtaining pure species from Quillajasaponin extracts.Furthermore, the structural identity of many Quillajasaponins remainsonly postulated. The discovery of new Quillajasaponins and relatedanalogs with potent adjuvant activity and low toxicity presents achallenge to the fields of chemical synthesis and medicine.

SUMMARY OF THE INVENTION

The present invention encompasses the recognition that the clinical useof QS-21 as an adjuvant is limited due to toxicity at higher doses, andthat QS-7, a related Quillajasaponin, is difficult to isolate in pureform. Moreover, synthetic access to QS-21, QS-7 and other triterpeneglycoside saponins is hindered by their structural complexity. Thepresent invention provides compounds that are analogs of QS-21 and QS-7.

In one aspect, the invention provides compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   is a single or double bond;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x),-   Y is CH₂, —O—, —NR—, or —NH—-   Z is hydrogen; a cyclic or acyclic, optionally substituted moiety    selected from the group consisting of acyl, aliphatic,    heteroaliphatic, aryl, arylalkyl, heterocyclyl, and heteroaryl; or a    carbohydrate domain having the structure:

-   -   wherein:

-   each occurrence of R¹ is R^(x) or a carbohydrate domain having the    structure:

-   wherein:-   each occurrence of a, b, and c is independently 0, 1, or 2;-   d is an integer from 1-5, wherein each d bracketed structure may be    the same or different; with the proviso that the d bracketed    structure represents a furanose or pyranose moiety, and the sum of b    and c is 1 or 2;-   R⁰ is hydrogen; an oxygen protecting group selected from the group    consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals,    ketals, esters, carbamates, and carbonates; or an optionally    substituted moiety selected from the group consisting of acyl, C₁₋₁₀    aliphatic, C₁₋₆ heteroaliphatic, 6-10-membered aryl, arylalkyl,    5-10-membered heteroaryl having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl    having 1-2 heteroatoms independently selected from the group    consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R^(a), R^(b), R^(c), and R^(d) is independently    hydrogen, halogen, OH, OR, OR^(x), NR₂, NHCOR, or an optionally    substituted group selected from acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur; 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R² is hydrogen, halogen, OH, OR, OC(O)R⁴, OC(O)OR⁴, OC(O)NHR⁴.    OC(O)NRR⁴, OC(O)SR⁴, NHC(O)R⁴, NRC(O)R⁴, NHC(O)OR⁴, NHC(O)NHR⁴,    NHC(O)NRR⁴, N(R⁴)₂, NHR⁴, NRR⁴, N₃, or an optionally substituted    group selected from C₁₋₁₀ aliphatic, C₁₋₆ heteroaliphatic,    6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R³ is hydrogen, halogen, CH₂OR¹, or an optionally substituted group    selected from the group consisting of acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur; 4-7-membered    heterocyclyl having 1-2 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur;-   R⁴ is

wherein X is —O— or —NR—; or

-   -   T-R^(z), wherein:

-   T is a covalent bond or a bivalent C₁₋₂₆ saturated or unsaturated,    straight or branched, aliphatic or heteroaliphatic chain; and

-   R^(z) is hydrogen, halogen, —OR, —OR^(x), —OR¹, —SR, —NR₂, —NC(O)OR,    or an optionally substituted group selected from acyl, arylalkyl,    heteroarylalkyl, C₁₋₆ aliphatic, 6-10-membered aryl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen and sulfur; or

-   two R⁴ on the same nitrogen atom are taken with the nitrogen to form    a 4-7-membered heterocyclic ring having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;

-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group selected from the group consisting of alkyl ethers,    benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates,    and carbonates;

-   R^(y) is —OH, —OR, or a carboxyl protecting group selected from the    group consisting of ester, amides, and hydrazides;

-   R^(s) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or:    -   two R^(x′) are taken together to form a 5-7-membered        heterocyclic ring having 1-2 heteroatoms independently selected        from the group consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₆ aliphatic, or C₁₋₆ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur; or: two R on the same nitrogen atom are taken    with the nitrogen to form a 4-7-membered heterocyclic ring having    1-2 heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur.

According to another aspect, the invention provides compounds of formulaIV:

-   wherein:-   is a single or double bond:-   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃ ³,    NHR, NH₂, SR, or NROR;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x);-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of ester, amides, and hydrazides;

R^(s) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or:    -   two R^(x′) are taken together to form a 5-7-membered        heterocyclic ring having 1-2 heteroatoms independently selected        from the group consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₁₂ aliphatic, or C₁₋₁₂ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur; and each occurrence of R^(x) is independently    hydrogen or an oxygen protecting group.

According to another aspect, inventive compounds have been shown to beuseful as adjuvants. Thus, in certain embodiments, vaccines are providedcomprising one or more bacterial, viral, protozoal, or tumor-associatedantigens, and one or more inventive compounds. In certain embodiments,one or more antigens are non-covalently associated with apharmaceutically acceptable excipient. In some embodiments, one or moreantigens are conjugated covalently to a pharmaceutically acceptableexcipient.

In another aspect, the present invention provides a method ofpotentiating an immune response to an antigen, comprising administeringto a subject a provided vaccine in an effective amount to potentiate theimmune response of said subject to said antigen.

In another aspect, the present invention provides methods of vaccinatinga subject, comprising administering a provided vaccine to said subject.In some embodiments, the subject is human. In some embodiments, thevaccine is administered orally. In other embodiments, the vaccine isadministered intramuscularly. In other embodiments, the vaccine isadministered subcutaneously. In certain embodiments, the amount ofadjuvant compound administered is 10-1000 μg. In certain embodiments,the amount of adjuvant compound administered is 500-1000 μg. In certainembodiments, the amount of adjuvant compound administered is 100-500 μg.In certain embodiments, the amount of adjuvant compound administered is50-250 μg. In certain embodiments, the amount of adjuvant compoundadministered is 50-500 μg. In certain embodiments, the amount ofadjuvant compound administered is 250-500 μg. The antigen to which thesubject is vaccinated may be a cancer, bacterial, viral, proatazoal, orself-antigen.

In another aspect, the invention provides pharmaceutical compositionscomprising compounds of the invention and pharmaceutically acceptableexcipients. In certain embodiments, the pharmaceutical composition is avaccine comprising an antigen and an inventive adjuvant.

In another aspect, the invention provides kits comprising pharmaceuticalcompositions of inventive compounds. In some embodiments, the kitscomprise prescribing information. In some embodiments, such kits includethe combination of an inventive adjuvant compound and anotherimmunotherapeutic agent (e.g.). The agents may be packaged separately ortogether. The kit optionally includes instructions for prescribing themedication. In certain embodiments, the kit includes multiple doses ofeach agent. The kit may include sufficient quantities of each componentto treat a subject for a week, two weeks, three weeks, four weeks, ormultiple months. In certain embodiments, the kit includes one cycle ofimmunotherapy. In certain embodiments, the kit includes a sufficientquantity of a pharmaceutical composition to immunize a subject againstan antigen long term.

In another aspect, the invention provides a method of using protectinggroups to isolate prosapogenins, the method comprising placingprotecting groups on a mixture of prosapogenins and then separating themixture by suitable means to isolate one or more prosapogenin compounds.In some embodiments, the method comprises the steps of:

(a) providing a mixture of prosapogenins of formula IV-a:

-   wherein:-   is a single or double bond;-   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃ ³,    NHR, NH₂, SR, or NROR;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x);-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of esters, amides, and hydrazides;-   R^(s1) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or:    -   two R^(x′) are taken together to form a 5-7-membered        heterocyclic ring having 1-2 heteroatoms independently selected        from the group consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₁₂ aliphatic, or C₁₋₁₂ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group selected from the group consisting of alkyl ethers,    benzyl ethers, silyl ethers, acetals, ketals, esters, and    carbonates;    (b) treating said compound of formula IV-a under suitable conditions    to form a mixture of prosapogenins of formula IV:

wherein each of

R^(y), Y′, V, and W is as defined for compounds of formula IV-a, R^(s)is as defined for compounds of formula I, and each occurrence of R^(x)is independently hydrogen or an oxygen protecting group selected fromthe group consisting of alkyl ethers, benzyl ethers, silyl ethers,acetals, ketals, esters, and carbonates;and(c) obtaining said compound IV by suitable physical means.

In some embodiments, the mixture of prosapogenins in step (a) isenriched one or more compounds of formula IV-a. In some embodiments, themixture of prosapogenins in step (b) is enriched one or more compoundsof formula N.

The present invention provides novel semi-synthetic methods forsynthesizing QS-7, QS-21, and related analogs, the method comprisingcoupling a triterpene compound with a compound comprising a saccharideto form a compound of formula I. In some embodiments, the methodcomprises the steps of:

(a) providing a compound of formula IV:

-   wherein:-   is a single or double bond;-   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃ ⁺,    NHR, NH₂, SR, or NROR;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x);-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of ester, amides, and hydrazides;-   R^(s) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or:    -   two R^(x′) are taken together to form a 5-7-membered        heterocyclic ring having 1-2 heteroatoms independently selected        from the group consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₁₂ aliphatic, or C₁₋₁₂ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group selected from the group consisting of alkyl ethers,    benzyl ethers, silyl ethers, acetals, ketals, esters, and    carbonates; (b) treating said compound of formula IV under suitable    conditions with a compound of formula V:

LG-Z   V

-   wherein:-   Z is hydrogen; a cyclic or acyclic, optionally substituted moiety    selected from the group consisting of acyl, aliphatic,    heteroaliphatic, aryl, arylalkyl, and heteroaryl; or a carbohydrate    domain having the structure:

-   wherein:-   each occurrence of R¹ is R^(x) or a carbohydrate domain having the    structure:

-   wherein:-   each occurrence of a, b, and c is independently 0, 1, or 2;-   d is an integer from 1-5, wherein each d bracketed structure may be    the same or different; with the proviso that the d bracketed    structure represents a furanose or pyranose moiety, and the sum of b    and c is 1 or 2;-   R⁰ is hydrogen, an oxygen protecting group selected from the group    consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals,    ketals, esters, carbamates, and carbonates; or an optionally    substituted moiety selected from the group consisting of acyl, C₁₋₁₀    aliphatic, C₁₋₆ heteroaliphatic, 6-10-membered aryl, arylalkyl,    5-10-membered heteroaryl having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl    having 1-2 heteroatoms independently selected from the group    consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R^(a), R^(b), R^(c), and R^(d) is independently    hydrogen, halogen, OH, OR, OR^(x), NR₂, NHCOR, or an optionally    substituted group selected from acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur; 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R² is hydrogen, halogen, OH, OR, OC(O)R⁴, OC(O)OR⁴, OC(O)NHR⁴,    OC(O)NRR⁴, OC(O)SR⁴, NHC(O)R⁴, NRC(O)R⁴, NHC(O)OR⁴, NHC(O)NHR⁴,    NHC(O)NRR⁴, N(R⁴)₂, NHR⁴, NRR⁴, N₃, or an optionally substituted    group selected from C₁₋₁₀ aliphatic, C₁₋₆ heteroaliphatic,    6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R³ is hydrogen, halogen, CH₂OR¹, or an optionally substituted group    selected from the group consisting of acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur; 4-7-membered    heterocyclyl having 1-2 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur;-   R⁴ is

wherein X is —O— or —NR—; or

-   -   T-R^(z), wherein:

-   T is a covalent bond or a bivalent C₁₋₂₆ saturated or unsaturated,    straight or branched, aliphatic or heteroaliphatic chain; and

-   R^(z) is hydrogen, halogen, —OR, —OR^(x), —OR¹, —SR, —NR₂, —NC(O)OR,    or an optionally substituted group selected from acyl, arylalkyl,    heteroarylalkyl, C₁₋₆ aliphatic, 6-10-membered aryl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen and sulfur; or

-   two R⁴ on the same nitrogen atom are taken with the nitrogen to form    a 4-7-membered heterocyclic ring having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;

-   each occurrence of R^(x) is as defined for compounds of formula IV;    and

-   LG is a suitable leaving group selected from the group consisting of    halogen, imidate, alkoxy, sulphonyloxy, optionally substituted    alkylsulphonyl, optionally substituted alkenylsulfonyl, optionally    substituted arylsulfonyl, and diazonium moieties;    -   to give a compound of formula I:

-   -   wherein each of        R^(x), R^(y), Z, V, and W is as defined for compounds of formula        IV or V, and Y is CH₂, —O—, —NR—, or —NH—.

Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “aliphatic” or “aliphatic group,” as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-12 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-6aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-5 aliphatic carbon atoms. In other embodiments, aliphatic groupscontain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic C₃-C₆ hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic, that has a single point of attachment to the rest of themolecule. Suitable aliphatic groups include, but are not limited to,linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynylgroups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₁₂ (or C₁₋₂₆, C₁₋₁₆, C₁₋₈) orsaturated or unsaturated, straight or branched, hydrocarbon chain,”refers to bivalent alkylene, alkenylene, and alkynylene chains that arestraight or branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkynylene” refers to a bivalent alkynyl group. A substitutedalkynylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “acyl,” used alone or a part of a larger moiety, refers togroups formed by removing a hydroxy group from a carboxylic acid.

The term “halogen” means F, Cl, Br, or I.

The terms “aralkyl” and “arylalkyl” are used interchangably and refer toalkyl groups in which a hydrogen atom has been replaced with an arylgroup. Such groups include, without limitation, benzyl, cinnamyl, anddihyrocinnamyl.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.”

In certain embodiments of the present invention, “aryl” refers to anaromatic ring system which includes, but not limited to, phenyl,biphenyl, naphthyl, anthracyl and the like, which may bear one or moresubstituents. Also included within the scope of the term “aryl,” as itis used herein, is a group in which an aromatic ring is fused to one ormore non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 n electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The terms “heteroaralkyl” and “heteroarylalkyl”refer to an alkyl group substituted by a heteroaryl moiety, wherein thealkyl and heteroaryl portions independently are optionally substituted.

The term “heteroaliphatic,” as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, or phosphorus. Heteroaliphatic groupsmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and include “heterocycle,” “hetercyclyl,”“heterocycloaliphatic,” or “heterocyclic” groups.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

In another aspect, the present invention provides “pharmaceuticallyacceptable” compositions, which comprise a therapeutically effectiveamount of one or more of the compounds described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail, the pharmaceuticalcompositions of the present invention may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to theskin, lungs, or oral cavity; intravaginally or intrarectally, forexample, as a pessary, cream or foam; sublingually; ocularly;transdermally; or nasally, pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, tertiary, or quaternary amine. Salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.Representative organic amines useful for the formation of base additionsalts include ethylamine, diethylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine and the like. (See, for example, Berge etal., supra).

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each stereocenter, Z and E double bond isomers, and Zand E conformational isomers. Therefore, single stereochemical isomersas well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.

Provided compounds may comprise one or more saccharide moieties. Unlessotherwise specified, both D- and L-configurations, and mixtures thereof,are within the scope of the invention. Unless otherwise specified, bothα- and β-linked embodiments, and mixtures thereof, are contemplated bythe present invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, chiralchromatography, or by derivation with a chiral auxiliary, where theresulting diastereomeric mixture is separated and the auxiliary groupcleaved to provide the pure desired enantiomers. Alternatively, wherethe molecule contains a basic functional group, such as amino, or anacidic functional group, such as carboxyl, diastereomeric salts areformed with an appropriate optically-active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is masked or blocked,permitting, if desired, a reaction to be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group is preferably selectively removable by readilyavailable, preferably non-toxic reagents that do not attack the otherfunctional groups; the protecting group forms a separable derivative(more preferably without the generation of new stereogenic centers); andthe protecting group will preferably have a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.By way of non-limiting example, hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), R-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described by Greene and Wuts (supra).

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄C(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)O—, Ph, —CH₂-(5-6-membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄straight or branched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen. C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable substituents on the aliphaticgroup of R^(†) are independently halogen, —R^(•), -(haloR^(•)), —OH,—OR^(•), —O(haloR^(•)), —CN, —C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•),—NR^(•) ₂, or —NO₂, wherein each R^(•) is unsubstituted or wherepreceded by “halo” is substituted only with one or more halogens, and isindependently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “enriched” as used herein refers to a mixture having anincreased proportion of one or more species. In some embodiments, themixture is “enriched” following a process that increases the proportionof one or more desired species in the mixture. In some embodiments, thedesired species comprise(s) greater than 10% of the mixture. In someembodiments, the desired species comprise(s) greater than 25% of themixture. In some embodiments, the desired species comprise(s) greaterthan 40% of the mixture. In some embodiments, the desired speciescomprise(s) greater than 60% of the mixture. In some embodiments, thedesired species comprise(s) greater than 75% of the mixture. In someembodiments, the desired species comprise(s) greater than 85% of themixture. In some embodiments, the desired species comprise(s) greaterthan 90% of the mixture. In some embodiments, the desired speciescomprise(s) greater than 95% of the mixture. Such proportions can bemeasured any number of ways, for example, as a molar ratio, volume tovolume, or weight to weight.

The term “pure” refers to compounds that are substantially free ofcompounds of related non-target structure or chemical precursors (whenchemically synthetized). This quality may be measured or expressed as“purity.” In some embodiments, a target compound has less than about30%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0.1% of non-target structures orchemical precursors. In certain embodiments, a pure compound of presentinvention is only one prosapogenin compound (i.e., separation of targetprosapogenin from other prosapogenins).

The term “carbohydrate” refers to a sugar or polymer of sugars. Theterms “saccharide”, “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose. (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a depicts the chemical structure of QS-7-Api.

FIG. 1 b depicts the chemical structures of QS-21-Api and QS-21-Xyl.Percentages correspond to the natural abundance of each isomer inisolated extracts of QS-21.

FIGS. 2 a-c show Anti-GD3 and Anti-KLH antibody titers after vaccinationwith GD3-KLH conjugate (10 μg) with adjuvants NQS-21, SQS-21-Mix,SQS-21-Api, or SQS-21-Xyl each at 20 μg doses: FIG. 2A IgM-GD3 AbTiters; FIG. 2B IgG-GD3 Ab Titers; and FIG. 2C IgG-KLH Ab Titers. Eachvalue represents median value of five mice (sera tested 7 days after3^(rd) and 4^(th) vaccination). NQS-21=naturally derived QS-21;SQS-21-Mix=synthetic QS-21.

FIG. 3 shows a comparison of cell surface reactivity against cell lineSK-Mel-28 between vaccinations with various SQS-adjuvants at 20 μgdoses.

FIG. 4 depicts a toxicity study in C57BL/6J Female Mice injected withGD3-KLH (10 μg) plus SQS-21-Mix, SQS-21-Api, or SQS-21-Xyl (20 μg each),compared to that with no adjuvant or natural NQS-21 (20 μg).

FIG. 5 shows the results on adjuvant activity of synthetic SQS-analoguesemploying GD3-KLH antigen. Antibody-titers are median values of groupsof five mice, wherein adjuvant dose is 10 μg. SQS-7=synthetic QS-7-Api.SQS-0101=compound I-9; SQS-0102=compound 1-10; SQS-0103=compound I-8.

FIGS. 6-14 show ¹H-NMR spectra of compounds described herein.

FIGS. 15 a-b are high-performance liquid chromatography traces of a 1:1mixture of natural and semisynthetic QS-7-Api.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The clinical success of anticancer and antimicrobial vaccines criticallydepends on the identification of, and access to, novel potent adjuvantswith attenuated toxicity. In this context, specific fractions fromextracts of the bark of Quillaja saponaria (QS) have proven to beexceedingly powerful adjuvants in immunotherapy. The QS-21 fraction(Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Immunol. 1991,146, 431-437), comprising isomeric forms of a complex triterpeneglycoside saponin (Soltysik, S.; Wu, J. Y.; Recchia, J.; Wheeler, D. A.;Newman, M. J.; Coughlin, R. T.; Kensil, C. R. Vaccine 1995, 13,1403-1410; Kensil, C. R. Crit. Rev. Ther. Drug Carrier Syst. 1996, 13,1-55), is currently the most promising immuno-potentiator (Kim, S. K.;Ragupathi, G.; Musselli, C.; Choi, S. J.; Park, Y. S.; Livingston, P. O.Vaccine 2000, 18, 597-603) in several antitumor (melanoma, breast, smallcell lung cancer, prostate) (Livingston, P. O.; Ragupathi, G. Hum.Vaccines 2006, 2, 137-143) and infectious-disease (HIV, malaria) vaccinetherapies (Sasaki, S.; Sumino, K.; Hamajima, K.; Fukushima, J.; Ishii,N.; Kawamoto, S.; Mohri, H.; Kensil, C. R.; Okuda, K. J. Virol. 1998,72, 4931-4939; Evans, T. G., et al. Vaccine 2001, 19, 2080-2091;Kashala, O., et al. Vaccine 2002, 20, 2263-2277; Carcaboso, A. M.;Hernandez, R. M.; Igartua, M.; Rosas, J. E.; Patarroyo, M. E.; Pedraz,J. L. Vaccine 2004, 22, 1423-1432). However, the tolerated dose of QS-21in cancer patients typically does not exceed 100 μg, above whichsignificant local erythema and systemic flu-like symptoms arise. On theother hand, QS-7, another QS extract fraction, was found not only topossess significant stand-alone adjuvant activity (Kensil, 1991; Kensil,1998, supra), but also to induce remarkable synergistic immune responseaugmentation (Kensil, C. A., U.S. Pat. No. 6,231,859) whenco-administered with QS-21, allowing for the administration of lessQS-21. Importantly, QS-7, unlike QS-21, exhibited negligible toxicity inmice. The present invention provides an efficient semi-synthetic methodof synthesizing analogs of QS-7 and QS-21, thereby significantlyreducing the number of synthetic steps required to access this potentclass of adjuvants.

Compounds

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. In some embodiments, provided compounds are analogs ofnaturally occurring triterpene glycoside saponins and intermediatesthereto. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed. Additionally,general principles of organic chemistry are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito: 1999,and March's Advanced Organic Chemistry, 5^(th) Ed., Ed.: Smith, M. B.and March, J., John Wiley & Sons, New York: 2001, the entire contents ofwhich are hereby incorporated by reference.

Description of Exemplary Compounds

In some embodiments, provided compounds are analogs of Quillajasaponins.In some embodiments, provided compounds are prosapogenins. In certainembodiments, provided compounds are analogs of QS-7 and QS-21 andpossess potent adjuvant activity.

In certain embodiments, the present invention provides a compound offormula I:

-   or a pharmaceutically acceptable salt thereof, wherein:-   is a single or double bond;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x),-   Y is CH₂, —O—, —NR—, or —NH—-   Z is hydrogen; a cyclic or acyclic, optionally substituted moiety    selected from the group consisting of acyl, aliphatic,    heteroaliphatic, aryl, arylalkyl, and heteroaryl; or a carbohydrate    domain having the structure:

-   -   wherein:

-   each occurrence of R¹ is R^(x) or a carbohydrate domain having the    structure:

-   wherein:-   each occurrence of a, b, and c is independently 0, 1, or 2,-   d is an integer from 1-5, wherein each d bracketed structure may be    the same or different; with the proviso that the d bracketed    structure represents a furanose or pyranose moiety, and the sum of b    and cis 1 or 2;-   R⁰ is hydrogen, an oxygen protecting group selected from the group    consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals,    ketals, esters, carbamates, and carbonates; or an optionally    substituted moiety selected from the group consisting of acyl, C₁₋₁₀    aliphatic, C₁₋₆ heteroaliphatic, 6-10-membered aryl, arylalkyl,    5-10-membered heteroaryl having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl    having 1-2 heteroatoms independently selected from the group    consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R^(a), R^(b), R^(c), and R^(d) is independently    hydrogen, halogen, OH, OR, OR^(x), NR₂, NHCOR, or an optionally    substituted group selected from acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur; 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R² is hydrogen, halogen, OH, OR, OR¹, OC(O)R⁴, OC(O)OR⁴, OC(O)NHR⁴,    OC(O)NRR⁴, OC(O)SR⁴, NHC(O)R⁴, NRC(O)R⁴, NHC(O)OR⁴, NHC(O)NHR⁴,    NHC(O)NRR⁴, N(R⁴)₂, NHR⁴, NRR⁴, N₃, or an optionally substituted    group selected from C₁₋₁₀ aliphatic, C₁₋₆ heteroaliphatic,    6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   R³ is hydrogen, halogen, CH₂OR¹, or an optionally substituted group    selected from the group consisting of acyl, C₁₋₁₀ aliphatic, C₁₋₆    heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur; 4-7-membered    heterocyclyl having 1-2 heteroatoms independently selected from the    group consisting of nitrogen, oxygen, and sulfur;-   R⁴ is

wherein X is —O— or —NR—; or

-   -   T-R^(z), wherein:

-   T is a covalent bond or a bivalent C₁₋₂₆ saturated or unsaturated,    straight or branched, aliphatic or heteroaliphatic chain; and

-   R^(z) is hydrogen, halogen, —OR, —OR^(x), —OR¹, —SR, —NR₂, —NC(O)OR,    or an optionally substituted group selected from acyl, arylalkyl,    heteroarylalkyl, C₁₋₆ aliphatic, 6-10-membered aryl, 5-10-membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2    heteroatoms independently selected from the group consisting of    nitrogen, oxygen and sulfur; or

-   two R⁴ on the same nitrogen atom are taken with the nitrogen to form    a 4-7-membered heterocyclic ring having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur

-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group selected from the group consisting of alkyl ethers,    benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates,    and carbonates.

-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of esters, amides, and hydrazides;

-   R^(s) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or:    -   two R^(x′) are taken together to form a 5-7-membered        heterocyclic ring having 1-2 heteroatoms independently selected        from the group consisting of nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₁₂ aliphatic, or C₁₋₁₂ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur; or: two R on the same nitrogen atom are taken    with the nitrogen to form a 4-7-membered heterocyclic ring having    1-2 heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur.

As defined above, W is Me, —CHO,

—CH₂OR^(x), or —C(O)OR^(y). In certain embodiments, W is methyl. Inother embodiments, W is —CHO. In certain embodiments, W is —CH₂OR^(x).In other embodiments, W is —C(O)OR^(y). In some embodiments, W is—CH₂OH. In other embodiments, W is —CH₂OBn. In other embodiments, W is—CH₂OSiEt₃. In certain embodiments, W is —C(O)OH. In other embodiments,W is —C(O)OBn.

In certain embodiments, V is —OR^(x). In some embodiments, V is —OH. Insome embodiments, V is hydrogen.

As defined above,

represents a single or double bond. It will be appreciated thatcompounds of formula I can be subjected to hydrogenation conditions(infra) that reduce the double bond to a single bond.

As defined above, Y is CH₂, —O—, —NR—, or —NH—. In certain embodiments,Y is CH₂. In certain embodiments, Y is —O—. In other embodiments, Y is—NR—. In some embodiments, Y is —NH—.

In certain embodiments, Z is hydrogen; a cyclic or acyclic, optionallysubstituted moiety selected from the group consisting of acyl,aliphatic, heteroaliphatic, aryl, arylalkyl, heterocyclyl, andheteroaryl.

In some embodiments, Z comprises a carbohydrate. In some embodiments, Zis not hydrogen. In other embodiments, Z is acyl.

In some embodiments, a Z comprises a linker group that separates acarbohydrate from Y. In some embodiments, the linker group is anoptionally substituted, straight or branched C₁₋₁₂ aliphatic orheteroaliphatic group. In some embodiments, the linker group is—(CH₂)_(k)—, wherein k is an integer between 1 and 10, inclusive.

In some embodiments, Z is an optionally substituted aliphatic group. Insome embodiments, Z is an optionally substituted C₁₋₃₀ aliphatic group.In some embodiments, Z is an optionally substituted C₁₋₂₀ aliphaticgroup. In some embodiments, Z is an optionally substituted C₁₋₁₆aliphatic group. In some embodiments, Z is an optionally substitutedC₁₋₁₂ aliphatic group. In some embodiments, Z is an optionallysubstituted C₁₋₁₀ aliphatic group. In some embodiments, Z is anoptionally substituted C₁₋₆ aliphatic group. In some embodiments, Z isan optionally substituted C₂₋₁₂ aliphatic group.

In some embodiments, Z is an optionally substituted heteroaliphaticgroup. In some embodiments, Z is an optionally substituted C₁₋₃₀heteroaliphatic group. In some embodiments, Z is an optionallysubstituted C₁₋₂₀ heteroaliphatic group. In some embodiments, Z is anoptionally substituted C₁₋₁₆ heteroaliphatic group. In some embodiments,Z is an optionally substituted C₁₋₁₂ heteroaliphatic group. In someembodiments, Z is an optionally substituted C₁₋₁₀ heteroaliphatic group.In some embodiments, Z is an optionally substituted C₁₋₆ heteroaliphaticgroup. In some embodiments, Z is an optionally substituted C₂₋₁₂heteroaliphatic group.

In certain embodiments, Z is an optionally substituted heteroaryl grouphaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, Z is is an optionally substituted5-12-membered heteroaryl group. In certain embodiments, Z is is anoptionally substituted 5-10-membered heteroaryl group. In certainembodiments, Z is is an optionally substituted 6-8-membered heteroarylgroup.

In certain embodiments, Z is an optionally substituted aryl group. Incertain embodiments, Z is is an optionally substituted 6-12-memberedaryl group. In certain embodiments, Z is is an optionally substituted6-10-membered aryl group. In certain embodiments, Z is is an optionallysubstituted 6-8-membered aryl group.

In some embodiments, Z is an optionally substituted heterocyclyl group.In certain embodiments, Z is an optionally substituted 4-7-memberedheterocyclyl group having 1-2 heteroatoms independently selected fromthe group consisting of nitrogen, oxygen, and sulfur.

In some embodiments, Z is an optionally substituted arylalkyl group. Insome embodiments, Z is an optionally substituted C₇₋₁₂ arylalkyl group.In some embodiments, Z is an optionally substituted C₇₋₁₀ arylalkylgroup. In some embodiments, Z is an optionally substituted C₇₋₈arylalkyl group.

In some embodiments, Z is a monosaccharide. In some embodiments, Z is anoligosaccharide. In certain embodiments, Z is a carbohydrate domainhaving the structure:

wherein each of R¹, R², and R³ is defined as described in classes andsubclasses above and herein.

In certain embodiments, Z has the structure:

wherein each of R⁰, R^(a), R^(b), R^(c), R^(d), a, b, c, and d isdefined as described in classes and subclasses above and herein.

As described above, the Z moiety is linked to the triterpene core via Y.In some embodiments, Z is a monosaccharide and is D-fucosyl. In someembodiments, Z is a monosaccharide and is L-fucosyl. In someembodiments, Z is a monosaccharide and is not fucosyl. In someembodiments, Z is a monosaccharide and is not β-D-fucosyl.

In some embodiments, Z is an oligosaccharide, and the carbohydratedomain directly attached to Y is fucosyl. In some embodiments, Z is anoligosaccharide, and the carbohydrate domain directly attached to Y isnot D-fucosyl. In some embodiments, Z is an oligosaccharide, and thecarbohydrate domain directly attached to Y is not β-D-fucosyl. In someembodiments, Z is an oligosaccharide, and the carbohydrate domaindirectly attached to Y is not α-D-fucosyl. In some embodiments, Z is anoligosaccharide, and the carbohydrate domain directly attached to Y isnot fucosyl.

In some embodiments, Z is an optionally substituted monosaccharide andis D-fucosyl. In some embodiments, Z is an optionally substitutedmonosaccharide and is L-fucosyl. In some embodiments, Z is an optionallysubstituted monosaccharide and is not β-D-fucosyl. In some embodiments,when a carbohydrate domain of Z is directly attached to Y, thecarbohydrate domain directly attached to Y is not fucosyl. In certainembodiments, when Y—Z is —OH, —OMe, or -Oallyl, at least seven R^(x)groups are silyl ethers. In some embodiments, Z and R^(x) are not allsimulataneously hydrogen or methyl. In some embodiments, Y—Z is not—OMe. In some embodiments, Y—Z is not —OH. In some embodiments, Y—Z isnot -Oallyl. In some embodiments, Y—Z is not —OH or —OMe if all R^(x)groups are simultaneously hydrogen or if at least four R^(x) groups aresimultaneously methyl.

In some embodiments, R^(y) is not a lipophilic group. In someembodiments, when a carbohydrate moiety of Z is non-acylated and allR^(x) are simulataneously hydrogen, the 3-O-glucuronic acid residue ofthe triterpene is not covalently attached, directly or indirectly, to acompound having a lipophilic domain, wherein the lipophilic domain isattached via the carboxylic acid carbon atom present on the3-O-glucuronic acid residue.

In certain embodiments, each occurrence of R^(y) is independently —OH.In certain embodiments, each occurrence of R^(y) is independently —OR.In certain embodiments, each occurrence of R^(y) is independently acarboxyl protecting group. Suitable carboxyl protecting groups are wellknown in the art and include those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference.

In some embodiments, each occurrence of R^(y), when taken with itsattached carbonyl group, independently comprises an ester. In someembodiments, each occurrence of R^(y), when taken with its attachedcarbonyl group, independently comprises an amide. In some embodiments,each occurrence of R^(y), when taken with its attached carbonyl group,independently comprises a hydrazide.

In some embodiments, each occurrence of R^(y) is independently —OBn. Inother embodiments, each occurrence of R^(y) is independently —OEt.

In certain embodiments, each occurrence of R^(x) is independentlyhydrogen. In certain embodiments, each occurrence of R^(x) isindependently a suitable hydroxyl protecting group. Suitable hydroxylprotecting groups are well known in the art and include those describedherein and by Greene (supra). In some embodiments, no more than fourR^(x) groups are simultaneously methyl.

In some embodiments, R^(s) is

In some embodiments, R^(s) is

In some embodiments, R^(s1) is

In some embodiments, R^(s1) is

In some embodiments, each occurrence of R^(x), when taken with itsattached oxygen atom, independently comprises a methyl ether, ethylether, benzyl ether, silyl ether, ester, acetal, ketal, or carbonate. Insome embodiments, R^(x) comprises a methyl ether. In some embodiments,R^(x) comprises a ethyl ether. In some embodiments, R^(x) comprises abenzyl ether. In some embodiments, R^(x) comprises a silyl ether. Insome embodiments, R^(x) comprises an ester. In some embodiments, R^(x)comprises an acetal. In some embodiments, R^(x) comprises a ketal. Insome embodiments, R^(x) comprises a carbonate.

In certain embodiments, R^(x) is methyl. In certain embodiments, R^(x)is ethyl. In certain embodiments, R^(x) is benzyl. In certainembodiments, R^(x) is SiR₃. In certain embodiments, R^(x) is SiMe₃. Incertain embodiments, R^(x) is TBS.

In certain embodiments, R^(x) is

In certain embodiments, R^(x) is

In certain embodiments, R^(x) is

In some embodiments, two —OR^(x) attached to adjacent carbon atoms on asaccharide ring are taken together to form a 5-7-membered heterocyclicring having 1-2 heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, two—OR^(x) attached to adjacent carbon atoms on a saccharide ring are takentogether to form a cyclic acetal protecting group. In some embodiments,two —OR^(x) attached to adjacent carbon atoms on a saccharide ring aretaken together to form a cyclic ketal protecting group.

In certain embodiments, each R^(x′) is independently hydrogen. Incertain embodiments, each Rx′ is independently an optionally substituted6-10-membered aryl group. In certain embodiments, each R^(x′) isindependently an optionally substituted C₁₋₆ aliphatic group. In certainembodiments, each R^(x′) is independently an optionally substituted C₁₋₆heteroaliphatic group having 1-2 heteroatoms independently selected fromthe group consisting of nitrogen, oxygen, and sulfur. In someembodiments, two R^(x′) are taken together to form a 5-7-memberedheterocyclic ring having 1-2 heteroatoms independently selected from thegroup consisting of nitrogen, oxygen, and sulfur.

In some embodiments, all R^(x) are hydrogen.

In certain embodiments, R¹ is a carbohydrate domain having thestructure:

-   -   wherein each of R⁰, R^(a), R^(b), R^(c), R^(d), a, b, c, and d        is defined as described in classes and subclasses above and        herein.

In some embodiments, a is 0. In some embodiments, a is 1.

In some embodiments, b is 0. In some embodiments, b is 1. In someembodiments, b is 2.

In some embodiments, c is 0. In some embodiments, c is 1. In someembodiments, c is 2.

In certain embodiments, the sum of b and c is 1. In certain embodiments,the sum of b and c is 2.

In certain embodiments, d is an integer from 1-7. In some embodiments, dis an integer from 1-5. In some embodiments, d is an integer from 1-4.In some embodiments, d is an integer from 1-2.

In certain embodiments, each d-bracketed structure is the same. Incertain embodiments, each d-bracketed structure is different. In certainembodiments, two or more d-bracketed structures are the same.

In some embodiments, one or more d-bracketed structures is a furanosemoiety. In some embodiments, one or more d-bracketed structure is apyranose moiety.

In some embodiments, R⁰ is hydrogen. In some embodiments, R⁰ is anoxygen protecting group selected from the group consisting of alkylethers, benzyl ethers, silyl ethers, acetals, ketals, esters,carbamates, and carbonates. In other embodiments, R⁰ is an optionallysubstituted moiety selected from the group consisting of acyl and C₁₋₁₀aliphatic.

In some embodiments, each occurrence of R^(a), R^(b), R^(c), and R^(d)is hydrogen. In some embodiments, each occurrence of R^(a), R^(b),R^(c), and R^(d) is —OH. In some embodiments, each occurrence of R^(a),R^(b), R^(c), and R^(d) is independently —OR. In some embodiments, eachoccurrence of R^(a), R^(b), R^(c), and R^(d) is independently —OR^(x).In some embodiments, each occurrence of R^(a), R^(b), R^(c), and R^(d)is independently an optionally substituted C₁₋₁₀ aliphatic group. Insome embodiments, each occurrence of R^(a), R^(b), R^(c), and R^(d) isindependently an optionally substituted C₁₋₆ heteroaliphatic group.

In some embodiments, each occurrence of R^(a), R^(b), R^(c), and R^(d)is —CH₂OH. In some embodiments, each occurrence of R^(a), R^(b), R^(c),and R^(d) is methyl.

As generally described above, in certain embodiments, R¹ is acarbohydrate domain. In some embodiments, R¹ is a monosaccharide. Insome embodiments, R¹ is an oligosaccharide. In certain embodiments, eachoccurrence of R¹ is independently selected from the group consisting of:

In certain embodiments, R² is hydrogen. In certain embodiments, R² ishalogen. In certain embodiments, R² is —OH. In certain embodiments, R²is OR. In certain embodiments, R² is —OC(O)R⁴. In certain embodiments,R² is —OC(O)OR⁴. In certain embodiments, R² is —OC(O)NHR⁴. In certainembodiments, R² is-OC(O)NRR⁴. In certain embodiments, R² is —OC(O)SR⁴.In certain embodiments, R² is —NHC(O)R⁴. In certain embodiments, R² is—NRC(O)R⁴. In certain embodiments, R² is —NHC(O)OR⁴. In certainembodiments, R² is —NHC(O)NHR⁴. In certain embodiments, R² is—NHC(O)NRR⁴. In certain embodiments, R² is —N(R⁴)₂. In certainembodiments, R² is —NHR⁴. In certain embodiments, R² is —NRR⁴. In someembodiments, R² is N₃.

In some embodiments, R² is an optionally substituted group selected fromC₁₋₃₀ aliphatic. In some embodiments, R² is an optionally substitutedgroup selected from C₁₋₂₀ aliphatic. In some embodiments, R² is anoptionally substituted group selected from C₁₋₁₀ aliphatic.

In some embodiments, R² is an optionally substituted group selected fromC₁₋₃₀ heteroaliphatic. In some embodiments, R² is an optionallysubstituted group selected from C₁₋₂₀ heteroaliphatic. In someembodiments, R² is an optionally substituted group selected from C₁₋₁₀heteroaliphatic. In some embodiments, R² is an optionally substitutedgroup selected from C₁₋₆ heteroaliphatic.

In some embodiments, R³ is hydrogen. In some embodiments, R³ is halogen.In some embodiments, R³ is —OH. In some embodiments, R³ is —OR. In someembodiments, R³ is —OR^(x). In some embodiments, R³ is an optionallysubstituted C₁₋₁₀ aliphatic group. In some embodiments, R³ is anoptionally substituted C₁₋₆ heteroaliphatic group. In some embodiments,R³ is not hydrogen. In some embodiments, R³ is not —OH.

In some embodiments, R³ is —CH₂OR. In some embodiments, R³ is —CH₂OH. Insome embodiments, R³ is methyl. In some embodiments, R³ is not methyl.In some embodiments, R³ is CH₂OR¹.

In some embodiments, R⁴ is

In some embodiments, X is —O—. In some embodiments, X is —NR—. In someembodiments, R⁴ is

In some embodiments, R⁴ is

In some embodiments, R⁴ is

In certain embodiments, R² is —NHC(O)R⁴; and R⁴ is

wherein p is an integer from 0 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴; and R⁴ is

wherein p is an integer from 0 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴; and R⁴ is

wherein p is an integer from 0 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴; and R⁴ is

wherein p is an integer from 0 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴; and R⁴ is

wherein p is an integer from 0 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴: and R⁴ is

wherein p is an integer from 1 to 12, inclusive. In certain embodiments,R² is —NHC(O)R⁴; and R⁴ is

In certain embodiments, two R⁴ on the same nitrogen atom are taken withthe nitrogen to form a 4-7-membered heterocyclic ring having 1-2heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur.

As described above, in certain embodiments, R⁴ is T-R^(z). In someembodiments, T is a covalent bond or a bivalent C₁₋₂₆ saturated orunsaturated, straight or branched, hydrocarbon chain, wherein one or twomethylene units of T are optionally and independently replaced by —O—,—S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O),—S(O)₂—, —N(R)SO₂—, or —SO₂N(R)—. In certain embodiments, T is acovalent bond or a bivalent C₁₋₁₆ saturated or unsaturated, straight orbranched, aliphatic or heteroaliphatic chain. In certain embodiments, Tis a covalent bond or a bivalent C₁₋₁₂ saturated or unsaturated,straight or branched, aliphatic or heteroaliphatic chain. In certainembodiments, T is a covalent bond or a bivalent C₁₋₈ saturated orunsaturated, straight or branched, aliphatic or heteroaliphatic chain.

In certain embodiments, -T- is selected from the group consisting of

In some embodiments, R^(z) is hydrogen. In some embodiments, R^(z) ishalogen. In certain embodiments, R^(z) is —NC(O)OR. In some embodiments,R^(z) is —OR. In some embodiments, R^(z) is —OR^(x). In someembodiments, R^(z) is —OR¹. In some embodiments, R^(z) is —NR₂. Incertain embodiments, R^(z) is an optionally substituted acyl group. Incertain embodiments, R is an optionally substituted arylalkyl group. Incertain embodiments, R^(z) is an optionally substituted heteroarylalkylgroup. In certain embodiments, R^(z) is an optionally substituted C₁₋₆aliphatic group. In certain embodiments, R^(z) is an optionallysubstituted 6-10-membered aryl group. In certain embodiments, R^(z) isan optionally substituted 5-10-membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfurgroup. In certain embodiments, R^(z) is an optionally substituted4-7-membered heterocyclyl having 1-2 heteroatoms independently selectedfrom the group consisting of nitrogen, oxygen and sulfur.

In certain embodiments, R^(z) is a monosaccharide. In certainembodiments, R^(z) is an oligosaccharide.

In certain embodiments, R^(z) is methyl. In certain embodiments, R^(z)is

In certain embodiments, R^(z) is

In some embodiments, each occurrence of R is independently hydrogen. Insome embodiments, each occurrence of R is independently an optionallysubstituted acyl group. In some embodiments, each occurrence of R isindependently an optionally substituted arylalkyl group. In someembodiments, each occurrence of R is independently an optionallysubstituted C₇₋₁₂ arylalkyl group. In some embodiments, each occurrenceof R is independently an optionally substituted 6-10-membered arylgroup. In some embodiments, each occurrence of R is independently anoptionally substituted C₁₋₁₂ aliphatic group. In some embodiments, eachoccurrence of R is independently an optionally substituted C₁₋₆aliphatic group. In some embodiments, each occurrence of R isindependently an optionally substituted C₁₋₆ heteroaliphatic grouphaving 1-2 heteroatoms independently selected from the group consistingof nitrogen, oxygen, and sulfur. In some embodiments, two R on the samenitrogen atom are taken with the nitrogen to form a 4-7-memberedheterocyclic ring having 1-2 heteroatoms independently selected from thegroup consisting of nitrogen, oxygen, and sulfur.

In certain embodiments, R⁵ and R⁶ are independently hydrogen, anoptionally substituted group selected from the group consisting of acyl,C₁₋₁₀ aliphatic, C₁₋₆ heteroaliphatic, 6-10-membered aryl, arylalkyl,5-10-membered heteroaryl having 1-4 heteroatoms independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur; 4-7-memberedheterocyclyl having 1-2 heteroatoms independently selected from thegroup consisting of nitrogen, oxygen, and sulfur. In certainembodiments, R⁵ and R⁶ are independently hydrogen. In some embodiments,R⁵ and R⁶ are independently —OH. In some embodiments, R⁵ and R⁶ areindependently —OR. In some embodiments, R⁵ and R⁶ are independently—OR^(x). In some embodiments, R⁵ and R⁶ are independently an optionallysubstituted C₁₋₁₀ aliphatic group. In some embodiments, R⁵ and R⁶ areindependently an optionally substituted C₁₋₆ heteroaliphatic group.

In some embodiments, R⁵ and R⁶ are independently —CH₂OR. In someembodiments, R⁵ and R⁶ are independently CH₂OH. In some embodiments, R⁵and R⁶ are independently methyl.

In some embodiments, each of R³, R⁵, and R⁶ is independently anoptionally substituted C₁₋₁₀ aliphatic group. In some embodiments, eachof R³, R⁵, and R⁶ is independently methyl. In some embodiments, each ofR³, R⁵, and R⁶ is independently —CH₂OR. In some embodiments, one or moreof R³, R⁵, and R⁶ is —CH₂OH. In some embodiments, each of R³, R⁵, and R⁶is —CH₂OH.

As described in further detail below, some materials used in thesynthesis of compounds of formula I may be commerically availableextracts derived from natural sources as mixtures of saponins. Suchextracts may contain saccharide moieties attached to the C3-position ofthe triterpene that differ from those depicted in formula I. Examples ofsaponins and prosapogenins that may be used according to the presentinvention include those derived from Glycyrrhizic acid, Hederasaponin C,β-Aescin, Helianthoside 2, Ginsenoside Rd, and Saponinum album, to namebut a few. All naturally-derived glycosylation variants of the C3position of the triterpene core are contemplated by the presentinvention.

In some embodiments, R^(s) of formula I is a xylose moiety, therebyproviding a compound of formula VII-a:

wherein each of R^(x), R^(y), W, V, Y and Z is defined as described inclasses and subclasses above and herein.

In some embodiments, R^(s) of formula I is a rhamnose moiety, therebyproviding a compound of formula VII-b:

wherein each of R^(x), R^(y), W, V, Y and Z is defined as described inclasses and subclasses above and herein.

In some embodiments, the triterpene core of formula I bears amonosaccharide at position C3. In some embodiments, amonosaccharide-substituted compound is of formula VIII:

wherein each of R^(x), R^(y), W, V, Y and Z is defined as described inclasses and subclasses above and herein.

In some embodiments, the triterpene core of formula I bears anoligosaccharide at position C3. In some embodiments, oligosaccharidewill contain rhamnose residues.

In certain embodiments, the triterpene core of formula I will beardisaccharides at position C3. In some embodiments, the disaccharide isgalactose-glucuronic acid. In some embodiments, adisaccharide-substituted compound is of formula IX:

wherein each of R^(x), R^(y), W, V, Y and Z is defined as described inclasses and subclasses above and herein.

In some embodiments, the triterpene core of formula I bears nosaccharide group at position C3, providing a compound of formula X:

wherein each of W, V, Y and Z is defined as described in classes andsubclasses above and herein.

Exemplary compounds of formula I are set forth in Table 1 below.

TABLE 1 Exemplary compounds of formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

In certain embodiments, a compound of formula I is not selected from:

It will be appreciated that it is not an object of the present inventionto claim compounds disclosed in the prior art that are the result ofisolation or degradation studies on naturally-occurring prosapongeninsor saponins.

As described above for compounds of formula I, in some embodiments, Y is—O—; Z is a carbohydrate domain having the structure:

wherein each occurrence of R¹ is R^(x) or a carbohydrate domaincomprising furanose or pyranose moieties; and R² is —NHC(O)R⁴.

Thus, according to one aspect, provided compounds are of formula II:

wherein each of R¹, R³, R⁴, R⁵, R^(x), R^(s), R^(y), V, and W is definedas described in classes and subclasses above and herein.

In some embodiments, provided compounds are of formula II-a:

wherein each of R¹, R³, R⁴, R⁵, R^(x), R^(s), R^(y), V, and W is definedas described in classes and subclasses above and herein.

In some embodiments, provided compounds are of formula II-b:

wherein each of R¹, R³, R⁴, R⁵, R^(x), R^(s), R^(y), V, and W is definedas described in classes and subclasses above and herein.

As described above for compounds of formula I, in some embodiments, Y is—O—; and Z is a carbohydrate domain having the structure:

wherein each occurrence of R¹ is R^(x) or a carbohydrate domaincomprising furanose or pyranose moieties.

Thus, according to one aspect, provided compounds are of formula III,III-a, III-b, III-c, III-d, III-e, III-f, or III-g:

wherein each of R¹, R², R³, R⁵, R⁶, R^(x), R^(s), R^(y), V, and W isdefined as described in classes and subclasses above and herein.

As described above for compounds of formula I, in certain embodiments,each occurrence of R^(x) is independently a suitable hydroxyl protectinggroup, Y is CH₂, —O—, —NR—, or —NH—; Z is hydrogen; a cyclic or acyclic,optionally substituted moiety selected from the group consisting ofacyl, aliphatic, heteroaliphatic, aryl, arylalkyl, and heteroaryl. Thus,according to another aspect, provided compounds are of formula IV:

-   wherein:-   is a single or double bond;-   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃ ⁺,    NHR, NH₂, SR, or NROR;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x);-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of ester, amides, and hydrazides;-   R^(s) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or: two R^(x′)    are taken together to form a 5-7-membered heterocyclic ring having    1-2 heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₁₂ aliphatic, or C₁₋₁₂ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group.

In some embodiments, at least one occurrence of R^(x) is an oxygenprotecting group. In some embodiments, at least two occurrences of R^(x)is an oxygen protecting group. In some embodiments, at least threeoccurrences of R^(x) is an oxygen protecting group. In some embodiments,at least four occurrences of R^(x) is an oxygen protecting group. Insome embodiments, all R^(x) are an oxygen protecting group. In someembodiments, each occurrence of R^(x) is independently an oxygenprotecting group. In certain embodiments, all R^(x) oxygen protectinggroups are the same. In some embodiments, at least one R^(x) oxygenprotecting group is different from the other R^(x) oxygen protectinggroups.

In some embodiments, Y′ is not —OMe. In some embodiments, Y′ is not —OH.In some embodiments, Y′ is not -Oallyl. In some embodiments, Y′ is not—OH or —OMe if all Rx groups are simultaneously hydrogen or if at leastfour R^(x) groups are simultaneously methyl.

In certain embodiments, at least one R^(x) group is not hydrogen. Incertain embodiments, at least one Rx group is not methyl. In certainembodiments, at least one R^(x) group is not hydrogen or methyl. Incertain embodiments, no R^(x) groups are hydrogen. In certainembodiments, no R^(x) groups are methyl. In certain embodiments, no Rxgroups are hydrogen or methyl.

In certain embodiments, provided compounds are of formula IV-b:

Exemplary compounds of formula IV are set forth in Table 1a below.

TABLE 1a Exemplary compounds of formula IV

Synthesis of Compounds

Quil-A (Accurate Chemical and Scientific Corporation, Westbury, N.Y.) isa commerically available semi-purified extract from Quillaja saonariawhich comprise a mixture of at least 50 distinct saponin species (vanSetten, D. C.; Vandewerken, G.; Zomer, G.; Kersten, G. F. A. RapidCommun. Mass Spectrom. 1995, 9, 660-666). Many of said saponin speciesinclude a triterpene-trisaccharide substructure as found inimmunologically-active Quillajasaponins such as QS-21 and QS-7:

It has been demonstrated (Guo, S., Kenne, L.; Lundgren, L. N., Rönnberg,B.; Sundquist, B. G. Phytochemistry, 1998, 48, 175-180) that exposure ofsaponin mixtures to base hydrolysis affords a mixture enriched withthree prosapongenins A, B, and C:

However, further use of this semi-pure hydrolyzed mixture ofprosapongenins is hindered by the fact that prosapongenins A and B,which differ only by an α-L-Rha vs. β-D-Xyl residue, are inseperable bysilica gel chromatography.

Other work has described isolation of highly pure or semi-pureprosapogenins and saponins (WO00/09075, U.S. Pat. Nos. 6,231,859,5,977,081, 6,080,725, 6,262,029; Higuchi, et al., Phytochemistry 1987,26, 229-235; Brown, F., Haahein, L R (eds), Dev Biol Stand., vol. 92:Brown, F. and Haahein, L R., Karger, Basel:1998, pp 41-47; Kensil, 1991,supra; van Setten, supra). However, these procedures are not efficientin terms of cost and labor, often requiring several rounds of silica geland/or HPLC purification in order to isolate the desired products.Furthermore, there is batch variability between commercially availableQS samples such that some batches contain no saponins withrhamnose-containing branched trisaccharides. None of the previouslydescribed isolation or degradation studies provides efficient access topure prosapogenins or sapogenins. For example, U.S. Pat. No. 6,231,859describes the isolation of 98% pure QS-21 following one silica gelchromatography and three or four additional rounds of HPLC run insequence. The final yield of QS-21 was 59 mg from 20 g of Quillajasaponaria extract (see Examples 1 and 2). A similar procedure for theisolation of QS-7 yielded 7 mg of purified QS-7 (final purity notreported) from a 20 g Quillaja saponaria extract (see Examples 1 and 4).Such yields and purity are not sufficient for large scale production ofpharmaceutical grade adjuvants.

In one aspect of the present invention, Applicant has unexpectedly founda strategy that allows for the facile separation of derivatizedprosapogenins A and B via silica gel chromatography. In one aspect, thehydroxyl groups on prosapogenins A and B are derivatized with a suitableprotecting group, as described herein, to afford derivatives that areseparable by silica gel chromatography. In some embodiments, allhydroxyl groups are derivatized with the same protecting group. In otherembodiments, different hydroxyl groups bear different protecting groups.In certain embodiments, the hydroxyl-protected prosapogenin A and Bderivatives also have a protecting group on one or both of theircarboxylic acid groups.

In certain embodiments, poly(silylation) of prosapogenins A and B,wherein all hydroxyl groups are converted to silyl ethers, gives amixture of poly(silylated) diacid prosapogenins A and B that are easilyseparable via silica gel chromatography. In some embodiments,poly(silylation) is used to afford nonakis(trialkylsilyl) ethers ofprosapogenins A and B. In some embodiments, poly(silylation) is used toafford nonakis(triethylsilyl) ethers of prosapogenins A and B. In someembodiments, poly(silylation) is used to afford nonakis(trimethylsilyl)ethers of prosapogenins A and B.

In other embodiments, the hydroxyl groups on prosapogenins A and B arederivatized as poly(benzyl) ethers to give a mixture of poly(benzyl)diacids of prosapogenins A and B that are easily seperable via silicagel chromatography. In some embodiments, poly(benzyl) etherification isused to afford nonakis(benzyl) ethers of prosapogenins A and B.

One of ordinary skill will appreciate that a number of suitableprotecting groups may be used, and that one or both carboxylic acidgroups may optionally be protected as well. In certain embodiments, theglucoronic acid group is selectively protected prior to derivitizing thehydroxyl groups of prosapogenins A and B. In some embodiments, theglucoronic acid group is selectively protected subsequent toderivitizing the hydroxyl groups of prosapogenins A and B.

Batch variability of commerically available Quillaja saponaria extractsmay result in hydrolyzed saponin mixtures containing prosapogenins otherthan A, B, and C. In certain embodiments, the hydrolyzed saponin mixturesubjected to the described protecting group strategy will be an enrichedmixture of prosapogenins A and B. In some embodiments, the saponinmixture will contain one or more other prosapogenins such asprosapogenin C. In certain embodiments, a preliminary round ofchromatography is used to render the saponin mixture enriched inprosapogenins A and B.

Other prosapongenins may be subjected to base hydrolysis and theprotecting group strategies described above and herein. Examples ofsaponins that may be derivatized with protecting groups according to thepresent invention include Glycyrrhizic acid, Hederasaponin C, β-Aescin,Helianthoside 2, Ginsenoside Rd, and Saponinum album, to name but a few.

In some embodiments, the mixture of prosapogenins will contain rhamnoseresidues. In some embodiments, the mixture of prosapogenins will notcontain rhamnose residues. In some embodiments, the mixture ofprosapogenins will be derived from Gypsoside A.

In certain embodiments, the mixture of prosapogenins will containdisaccharides. In some embodiments, the disaccharide isgalactose-glucuronic acid.

As described herein, prosapogenins bearing protecting groups inaccordance with the present invention may be separated and isolated fromone another by suitable physical means. The term “separated by suitablephysical means” refers to methods of separating mixtures ofprosapogenins or saponins. Such methods are well known in the art andinclude preferential crystallization, chromatography, and trituration,among others. One of ordinary skill in the art will recognize that suchmethods may allow for the separation and isolation of both major andminor constituents.

In some embodiments, suitable protecting groups will render providedcompounds crystalline. In certain embodiments, preferentialcrystallization is used to separate provided compounds.

It will be appreciated that chromatography steps aimed at separatingderivatives of prosapogenins A and B may be carried out according tomethods known in the art. In certain embodiments, chromatography iscarried out on derivatives of prosapogenins A and B wherein all hydroxylgroups bear a suitable protecting group. In some embodiments,chromatography is carried out on poly(benzyl) diacids of prosapogenins Aand B. In some embodiments, chromatography is carried out on poly(silyl)ether diacids of prosapogenins A and B. In some embodiments,chromatography is carried out on poly(benzyl) ethers of prosapogenins Aand B wherein one or both carboxylic acid groups bears a protectinggroup. In some embodiments, chromatography is carried out on poly(silyl)ethers of prosapogenins A and B wherein one or both carboxylic acidgroups bears a protecting group.

In certain embodiments, the chromatography is gravity silica gelchromatography. In certain embodiments, the chromatography is flashsilica gel chromatography. In certain embodiments, the chromatography isgravity alumina gel chromatography. In certain embodiments, thechromatography is flash alumina gel chromatography. In certainembodiments, the chromatography is high pressure liquid chromatography(HPLC).

In some embodiments, separation by suitable physical means yieldsprovides prosapogenin compounds of >70% purity. In some embodiments,separation by suitable physical means yields provides prosapogenincompounds of >80% purity. In some embodiments, separation by suitablephysical means yields provides prosapogenin compounds of >90% purity. Insome embodiments, separation by suitable physical means yields providesprosapogenin compounds of >95% purity. In some embodiments, separationby suitable physical means yields provides prosapogenin compoundsof >98% purity. In some embodiments, separation by suitable physicalmeans yields provides prosapogenin compounds of >99% purity. In someembodiments, separation by suitable physical means yields providesprosapogenin compounds of >99.5% purity. In some embodiments, separationby suitable physical means yields provides prosapogenin compoundsof >99.9% purity.

In certain embodiments, provided compounds of formula I have >80%purity. In some embodiments, provided compounds of formula I have >90%purity. In some embodiments, provided compounds of formula I have >95%purity. In some embodiments, provided compounds of formula I have >98%purity. In some embodiments, provided compounds of formula I have >99%purity. In some embodiments, provided compounds of formula I have >99.5%purity. In some embodiments, provided compounds of formula I have >99.9%purity.

Thus, according to another aspect, the invention provides a method ofusing protecting groups to isolate prosapogenins, the method comprisingthe steps of:

(a) providing a mixture of prosapogenins of formula IV-a:

-   wherein:-   is a single or double bond;-   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃ ⁺,    NHR, NH₂, SR, or NROR;-   W is Me, —CHO,

—CH₂OR^(x), or —C(O)R^(y);

-   V is hydrogen or —OR^(x);-   R^(y) is —OH, or a carboxyl protecting group selected from the group    consisting of ester, amides, and hydrazides;-   R^(s1) is

-   each occurrence of R^(x′) is independently an optionally substituted    group selected from 6-10-membered aryl, C₁₋₆ aliphatic, or C₁₋₆    heteroaliphatic having 1-2 heteroatoms independently selected from    the group consisting of nitrogen, oxygen, and sulfur; or: two R^(x′)    are taken together to form a 5-7-membered heterocyclic ring having    1-2 heteroatoms independently selected from the group consisting of    nitrogen, oxygen, and sulfur;-   each occurrence of R is independently hydrogen, an optionally    substituted group selected from acyl, arylalkyl, 6-10-membered aryl,    C₁₋₆ aliphatic, or C₁₋₆ heteroaliphatic having 1-2 heteroatoms    independently selected from the group consisting of nitrogen,    oxygen, and sulfur;-   each occurrence of R^(x) is independently hydrogen or an oxygen    protecting group selected from the group consisting of alkyl ethers,    benzyl ethers, silyl ethers, acetals, ketals, esters, and    carbonates;    (b) treating said compound of formula IV-a under suitable conditions    to form a mixture of prosapogenins of formula IV:

wherein each of

R^(y), Y′, V, and W is as defined for compounds of formula IV-a, R^(s)is as defined for compounds of formula I, and each occurrence of R^(x)is independently hydrogen or an oxygen protecting group selected fromthe group consisting of alkyl ethers, benzyl ethers, silyl ethers,acetals, ketals, esters, and carbonates;and(c) obtaining said compound IV by suitable physical means.

As described above, the present invention provides methods of preparingcompounds of formula I. In some embodiments, the R^(x) and R^(y) groupsof provided compounds are suitable protecting groups. Without wishing tobe bound by any particular theory, it is believed that the presence ofsaid protecting groups on provided compounds of formula N is useful inthe reaction of compounds of formula IV with a compound of formula V toform a compound of formula I. As depicted below in Scheme 1, a compoundof formula IV may be reacted under suitable conditions with a compoundof formula V to provide a compound of formula I.

wherein each of

, R^(x), R^(y), V, and W is defined as described in classes andsubclasses above and herein;

-   -   Y′ is hydrogen, halogen, alkyl, aryl, OR, OR^(y), OH, NR₂, NR₃,        NHR, NH₂, SR, or NROR;    -   Z is hydrogen; a cyclic or acyclic, optionally substituted        moiety selected from the group consisting of acyl, aliphatic,        heteroaliphatic, aryl, arylalkyl, and heteroaryl; or a        carbohydrate domain having the structure:

-   -   wherein:    -   each occurrence of R¹ is R^(x) or a carbohydrate domain having        the structure:

-   -   -   wherein:        -   each occurrence of a, b, and c is independently 0, 1, or 2;        -   d is an integer from 1-5, wherein each d bracketed structure            may be the same or different; with the proviso that the d            bracketed structure represents a furanose or pyranose            moiety, and the sum of b and c is 1 or 2;        -   R⁰ is hydrogen, an oxygen protecting group selected from the            group consisting of alkyl ethers, benzyl ethers, silyl            ethers, acetals, ketals, esters, carbamates, and carbonates;            or an optionally substituted moiety selected from the group            consisting of acyl, C₁₋₁₀ aliphatic, C₁₋₆ heteroaliphatic,            6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl            having 1-4 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2            heteroatoms independently selected from the group consisting            of nitrogen, oxygen, and sulfur;

    -   each occurrence of R^(a), R^(b), R^(c), and R^(d) is        independently hydrogen, halogen, OH, OR, OR^(x), NR₂, NHCOR, or        an optionally substituted group selected from acyl, C₁₋₁₀        aliphatic, C₁₋₆ heteroaliphatic, 6-10-membered aryl, arylalkyl,        5-10-membered heteroaryl having 1-4 heteroatoms independently        selected from nitrogen, oxygen, or sulfur; 4-7-membered        heterocyclyl having 1-2 heteroatoms independently selected from        the group consisting of nitrogen, oxygen, and sulfur.

One of ordinary skill in the art will appreciate that provided compoundscan be subjected to reductive conditions (i.e., LiAlH₄, NaBH₄, AlH₃,NaBH₃(OAc), Zn(BH₄)₂, Et₃SiH, and others described in March, supra) totransform the aldehyde moiety into an alcohol or methyl group. Providedcompounds can also be subjected to oxidative conditions (i.e., MnO₄ ⁻,chromic acid, bromine, Oxone®, silver oxide, and others described inMarch, supra) to transform the aldehyde moiety into a carboxyl group.Such hydroxyl or carboxyl groups can be protected with suitableprotecting groups as defined above and herein.

Thus, in certain embodiments, W is Me, —CHO,

—CH₂OR^(x), or —C(O)OR^(y). In certain embodiments, W is methyl. Inother embodiments, W is —CHO. In certain embodiments, W is —CH₂OR^(x).In other embodiments, W is —C(O)OR. In some embodiments, W is —CH₂OH. Inother embodiments, W is —CH₂OBn. In other embodiments, W is —CH₂OSiEt₃.In certain embodiments, W is —C(O)OH. In other embodiments, W is—C(O)OBn.

In certain embodiments, V is —OR^(x). In some embodiments, V is —OH. Insome embodiments, V is hydrogen.

As defined above,

represents a single or double bond. It will be appreciated thatcompounds of formula IV can be subjected to hydrogenation conditions(i.e., Raney-Ni, Pd/C, NaBH₄, reduced nickel, Adams' catalyst, zincoxide, Wilkinson's catalyst, and others described in March, supra) thatreduce the double bond to a single bond.

It will be appreciated that the C28 carboxyl group may be transformedinto other carbonyl functional groups. In some embodiments, the carboxylgroup is reduced to an aldehyde. In other embodiments, the carboxylgroup is converted into a Weinreb amide. In other embodiments, thecarboxyl group is converted into an amide. In other embodiments, thecarboxyl group is converted into an ester.

As defined above, Y is CH₂, —O—, —NR—, or —NH—. In certain embodiments,Y is CH₂. In certain embodiments, Y is —O—. In other embodiments, Y is—NR—. In some embodiments, Y is —NH—.

The present invention encompasses the recognition that judiciousselection of protecting groups on provided compounds of formula IVallows for the derivation of the —C(O)Y′ group attached to C28. Incertain embodiments, —C(O)Y′ is a ketone. In other embodiments, —C(O)Y′is an amide. In some embodiments, —C(O)Y′ is an ester.

The LG group of formula V is a suitable leaving group. One of ordinaryskill in the art will appreciate that a variety of suitable leavinggroups LG can be used to facilitate the reaction described in step S-1,and all such suitable leaving groups are contemplated by the presentinvention. A suitable leaving group is a chemical group that is readilydisplaced by a desired incoming chemical moiety. Suitable leaving groupsare well known in the art, e.g., see, March, supra. Such leaving groupsinclude, but are not limited to, halogen, alkoxy, sulphonyloxy,optionally substituted alkylsulphonyl, optionally substitutedalkenylsulfonyl, optionally substituted arylsulfonyl, and diazoniummoieties. Examples of some suitable leaving groups include chloro, iodo,bromo, fluoro, methanesulfonyl (mesyl), tosyl, triflate,nitro-phenylsulfonyl (nosyl), and bromo-phenylsulfonyl (brosyl).Additional leaving groups are described herein.

In certain embodiments, a compound of formula V is a monosacchide oroligosaccharide that may act as a glycosylation donor. Thus, accordingto another aspect of the invention, provided compounds are of formulaeVI or VI′:

wherein each of R¹, R², and R³ is defined as described in classes andsubclasses above and herein; and LG¹ is a suitable leaving group.

As depicted in Scheme 2, a compound of formula IV may be reacted undersuitable conditions with a compound of formula VI to give a compound offormula I-A:

wherein each of

, R^(x), R^(y), R^(s), V, W, Y′, Y, R¹, R², R³, and LG¹ is defined asdescribed in classes and subclasses above and herein.

The LG¹ group of compounds of formulae VI and VI′ is a glycoside donorleaving group, as defined and described herein. One of ordinary skill inthe art will appreciate that a variety of suitable leaving groups LG¹can be used to facilitate the reaction described, and all such suitableleaving groups are contemplated by the present invention.

In some embodiments, LG¹ is monovalent. In other embodiments, LG¹ isdivalent. In certain embodiments, the LG group of formula V is halogen,thioalkyl, thioaryl, thioheteroaryl, thiocyano, O-acyl, orthoester,O-carbonate, S-carbonate, trichloroimidate, 4-pentenyl, phosphate,O-sulfonyl, O-silyl, hydroxyl, diazirine, or arylseleno.

As described above, in certain embodiments the LG¹ group is halogen. Insome embodiments, LG¹ is Br. In some embodiments, LG¹ is Cl. In someembodiments, LG¹ is F.

In some embodiments, LG¹ is thioalkyl. In some embodiments, LG¹ is —SEt.In some embodiments, LG¹ is —SMe.

In some embodiments, LG¹ is thioaryl. In some embodiments, LG¹ is —SPh.

In some embodiments, LG¹ is thioheteroaryl. In some embodiments, LG¹ isthiopyridinyl (-SPy). In some embodiments, LG¹ is

In some embodiments, LG¹ is

In some embodiments, LG¹ is O-acyl. In some embodiments, LG¹ is —OAc. Insome embodiments, LG¹ is —OC(O)CH₂Br. In some embodiments, LG¹ is —OBz.In some embodiments, LG¹ is —OC(O)C₆H₄-p-NO₂. In some embodiments, LG¹is —OC(O)Py.

In some embodiments, the LG¹ group maybe taken together with anotherpart of Z to form a cyclic moiety. In certain embodiments, the takingtogether of LG¹ with another part of Z forms an ortho ester orderivative thereof. In certain embodiments, the LG¹ group comprises atert-butyl ortho ester. In some embodiments, the LG¹ group comprises a1-cyanoethylidene. In some embodiments, the LG¹ group comprises a(p-methylphenyl)thioethylidene. In some embodiments, the LG¹ groupcomprises an ethylthioethylidene. In some embodiments, the LG¹ groupcomprises an [N-(1-phenylethylidene)amino]oxyl-2,2-dimethylpropylidene.In some embodiments, the LG¹ group comprises a cyclic thiocarbonate. Insome embodiments, the LG¹ group comprises a diazirine.

In some embodiments, LG¹ is O-carbonate. In some embodiments, LG¹ isO-xanthate. In some embodiments, LG¹ is —OC S SMe. In some embodiments,LG¹ is

In some embodiments, LG¹ is

In some embodiments, LG¹ is —SC(S)—OEt. In some embodiments, LG¹ is

In certain embodiments, LG¹ is trichloroimidate. In some embodiments,LG¹ is —OC(NH)CCl₃.

In certain embodiments, LG¹ is 4-pentenyl. In some embodiments, LG¹ is—O(CH₂)₃CHCH₂.

In certain embodiments, LG¹ comprises a phosphate. In some embodiments,LG¹ comprises a diphenyl phosphate. In some embodiments, LG¹ comprises adiphenylphosphineimidate. In some embodiments, LG¹ comprises aphosphoroamidate. In some embodiments, LG¹ comprises aphosphorodiamidimidothioate. In some embodiments, LG¹ comprises adimethylphosphinothioate.

In certain embodiments, LG¹ is O-sulfonyl. In some embodiments, LG¹ is—OTs. In some embodiments, LG¹ is —OMs. In some embodiments, LG¹ is—OTf.

In some embodiments, LG¹ is O-silyl. In some embodiments, LG¹ is —OTMS.In some embodiments, LG¹ is —OSiEt₃. In some embodiments, LG¹ is —OTBS.

In certain embodiments, LG¹ is hydroxyl.

In certain embodiments, LG¹ is α-linked compound of formula VI. Incertain embodiments, LG¹ is β-linked compound of formula VI.

In certain embodiments, LG¹ is n-alkenyl.

General methods and reagents for carrying out glycosylation reactionsare described by Toshima, K. and Tatsuta, K., Chem. Rev. 1993, 93,1503-1531, the entire contents of which is hereby incorporated byreference.

In certain embodiments, compounds of formula I are provided byconjugating an oligosaccharide of formulae VI or VI′ with a compound offormula IV as described for step S-1. In some embodiments, the entireoligosaccharide is prepared as a compound of formula VI prior to stepS-1. In other embodiments, a monosaccharide of formula V1 is conjugatedin step S-1 to a compound of formula IV, and the resultingtriterpene-saccharide conjugate is subjected to further glycosylationreactions to provide a compound of formula I. In some embodiments,protecting group strategies are employed that allow for selectiveglycosylation reactions to occur in the assembly of the final triterpeneoligosaccharide compound of formula I.

Thus, in another aspect, the present invention provides compounds offormulae VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, VI-7, VI-8, VI-9, VI-10,and VI-11:

wherein each of R², R³, R⁴, R⁵, R⁶, R^(x), and LG¹ is defined asdescribed in classes and subclasses above and herein.

In certain embodiments, compounds of formula VI are monosaccharide andD-fucosyl. In some embodiments, compounds of formula VI aremonosaccharide and L-fucosyl. In some embodiments, compounds of formulaVI are monosaccharide and are not fucosyl. In some embodiments,compounds of formula VI are monosaccharide and are not D-fucosyl. Insome embodiments, compounds of formula VI are monosaccharide and are notβ-D-fucosyl.

In some embodiments, compounds of formula VI are oligosaccharide, andthe carbohydrate domain directly attached to Y is fucosyl. In someembodiments, compounds of formula VI are oligosaccharide, and thecarbohydrate domain directly attached to Y is not D-fucosyl. In someembodiments, compounds of formula VI are oligosaccharide, and thecarbohydrate domain directly attached to Y is not β-D-fucosyl. In someembodiments, compounds of formula VI are oligosaccharide, and thecarbohydrate domain directly attached to Y is not α-D-fucosyl. In someembodiments, compounds of formula VI are oligosaccharide, and thecarbohydrate domain directly attached to Y is not fucosyl.

In some embodiments, compounds of formula VI are optionally substitutedmonosaccharide and D-fucosyl. In some embodiments, compounds of formulaVI are optionally substituted monosaccharide and L-fucosyl. In someembodiments, compounds of formula VI are optionally substitutedmonosaccharide and not fucosyl. In some embodiments, compounds offormula VI are optionally substituted monosaccharide and notβ-D-fucosyl.

Exemplary compounds of formula VI are set forth in Table 2.

TABLE 2 Exemplary compounds of formula VI

In certain embodiments, the present compounds are generally preparedaccording to Scheme 3 set forth below:

wherein each of R⁶ and R^(x) is defined as described in classes andsubclasses above and herein.

In some embodiments, the present compounds are generally preparedaccording to Scheme 4 set forth below:

wherein each of R³ and R^(x) is defined as described in classes andsubclasses above and herein.

In some embodiments, the present compounds are generally preparedaccording to Scheme 5 set forth below:

wherein each of R³, R⁶, and R^(x) is defined as described in classes andsubclasses above and herein.

In some embodiments, the present compounds are generally preparedaccording to Scheme 6 set forth below:

wherein each of R¹, R³, R⁵, R⁶, LG¹, and R^(x) is defined as describedin classes and subclasses above and herein.

In each of the synthetic steps depicted in Schemes 3-6, one of ordinaryskill will recognize that a variety of suitable protecting groups may beused. Orthogonal protecting group strategies are well known in the artand may be used to selectively protect and deprotect saccharide hydroxylgroups. It will be appreciated that a variety of suitable leaving groupsLG¹ may also be employed, as described above, to carry out glycosylationstep S-9.

In certain embodiments, step S-1 involves the addition of a nucleophileto a compound of formula IV. In some embodiments, the reaction between acompound of formula IV and a nucleophile is carried out using suitableesterification conditions. The term “suitable esterificationconditions,” as used herein, refers to the catalyzed or uncatalyzedesterification or transesterification between an oxygen nucleophile andan ester or carboxylic acid. In some embodiments, the conditionscomprise the addition or one or more bases. In some embodiments, thebase is an amine. In some embodiments, an additional promoter ofesterification may be used such as DMAP or EDC.

In some embodiments, the reaction between a compound of formula IV and anucleophile is carried out using suitable peptide bond formingconditions. Suitable peptide coupling conditions are well known in theart and include those described in detail in Han et al., Tetrahedron,60, 2447-67 (2004), the entirety of which is hereby incorporated byreference. In certain embodiments, the peptide coupling conditionsinclude the addition of HOBt, DMAP, BOP, HBTU, HATU, BOMI, DCC, EDC,IBCF, or a combination thereof.

In some embodiments, the nucleophile is carbon-based, such as an alkylmetal species. In some embodiments, the nucleophile is a Grignardreagent. In some embodiments, the nucleophile is an organolithium. Insome embodiments, the nucleophile is an organoborane. In someembodiments, the nucleophile is an organotin. In some embodiments, thenucleophile is an enol.

Uses

Compounds of formulae I, II, III, or IV may be used as adjuvants or toenhance the cellular uptake of toxins. The inventive compounds may beparticularly useful in the treatment or prevention of neoplasms or otherproliferative diseases in vivo. However, inventive compounds describedabove may also be used in vitro for research or clinical purposes.

Adjuvants

Most protein and glycoprotein antigens are poorly immunogenic ornon-immunogenic when administered alone. Strong adaptive immuneresponses to such antigens often requires the use of adjuvants. Immuneadjuvants are substances that, when administered to a subject, increasethe immune response to an antigen or enhance certain activities of cellsfrom the immune system. An adjuvant may also allow the use of a lowerdose of antigen to achieve a useful immune response in a subject.

Common adjuvants include alum, Freund's adjuvant (an oil-in-wateremulsion with dead mycobacteria), Freund's adjuvant with MDP (anoil-in-water emulsion with muramyldipeptide, MDP, a constituent ofmycobacteria), alum plus Bordetella pertussis (aluminum hydroxide gelwith killed B. pertussis). Such adjuvants are thought to act by delayingthe release of antigens and enhancing uptake by macrophages. Immunestimulatory complexes (ISCOMs) such as Quil-A (a Quillaja saponinextract) are open cage-like complexes typically with a diameter of about40 nm that are built up by cholesterol, lipid, immunogen, and saponin.ISCOMs deliver antigen to the cytosol, and have been demonstrated topromote antibody response and induction of T helper cell as well ascytotoxic T lymphocyte responses in a variety of experimental animalmodels.

Various studies have raised the concern of potential toxicity associatedwith saponin-based adjuvants. Fractionation experiments testing themajor components of Quil-A showed that QS-21 had low toxicity and QS-7showed no lethality at the doses tested in CD-1 mice intradermally. QS-7showed no hemolytic activity at levels up to 200 μg/mL of saponin(Kensil et al., 1991, supra).

In humans, QS-21 has displayed both local and systemic toxicity. Maximumdoses for healthy patients are typically ≤50 μg, and ≤100 μg for cancerpatients. As mentioned above, QS-7 has been found not only to possesssignificant stand-alone adjuvant activity, but also to induce remarkablesynergistic immune response augmentation. Unfortunately, QS-7 has provendifficult to isolate in clinically useful quantities.

The present invention encompasses the recognition that synthetic accessto and structural modification of QS-7 and related Quillajasaponins mayafford compounds with high adjuvant potency and low toxicity.

Enhanced Uptake of Toxins

Saponins have been shown to exhibit cell membrane-permeabilizingproperties, and have been investigated for their therapeutic potential.In some cases, saponins have virtually no effect alone, but when used incombination with another drug will significantly amplify the effects ofthe other drug. One example of such a combination effect is withginsenoside and cis-diaminedichloroplatinum(II) (Nakata, H., et al., JpnJ Cancer Res. 1998, 89, 733-40). Therefore, saponins have potentialutility in combination therapies with antitumor drugs for cancertreatment. Saponinum album from Gypsophila paniculata L. has beendescribed to enhance the cytotoxicity of a chimeric toxin in cellculture, even at nonpermeabilizing concentrations.

In certain embodiments, provided compounds may be used to enhance theuptake of other cytotoxic agents.

Vaccines

Compositions of the invention are useful as vaccines to induce activeimmunity towards antigens in subjects. Any animal that may experiencethe beneficial effects of the compositions of the present inventionwithin the scope of subjects that may be treated. In some embodiments,the subjects are mammals. In some embodiments, the subjects are humans.

The vaccines of the present invention may be used to confer resistanceto infection or cancer by either passive or active immunization. Whenthe vaccines of the present invention are used to confer resistancethrough active immunization, a vaccine of the present invention isadministered to an animal to elicit a protective immune response whicheither prevents or attenuates a proliferative or infectious disease.When the vaccines of the present invention are used to confer resistanceto infection through passive immunization, the vaccine is provided to ahost animal (e.g., human, dog, or mouse), and the antisera elicited bythis vaccine is recovered and directly provided to a recipient suspectedof having an infection or disease or exposed to a causative organism.

The present invention thus concerns and provides a means for preventingor attenuating a proliferative disease resulting from organisms or tumorcells which have antigens that are recognized and bound by antiseraproduced in response to the immunogenic polypeptides included invaccines of the present invention. As used herein, a vaccine is said toprevent or attenuate a disease if its administration to an animalresults either in the total or partial attenuation (i.e., suppression)of a symptom or condition of the disease, or in the total or partialimmunity of the animal to the disease.

The administration of the vaccine (or the antisera which it elicits) maybe for either a “prophylactic” or “therapeutic” purpose. When providedprophylactically, the vaccine(s) are provided in advance of any symptomsof proliferative disease. The prophylactic administration of thevaccine(s) serves to prevent or attenuate any subsequent presentation ofthe disease. When provided therapeutically, the vaccine(s) is providedupon or after the detection of symptoms which indicate that an animalmay be infected with a pathogen or have a certain cancer. Thetherapeutic administration of the vaccine(s) serves to attenuate anyactual disease presentation. Thus, the vaccines may be provided eitherprior to the onset of disease proliferation (so as to prevent orattenuate an anticipated infection or cancer) or after the initiation ofan actual proliferation.

Thus, in one aspect the present invention provides vaccines comprisingone or more bacterial, viral, protozoal, or tumor-related antigens incombination with one or more inventive compounds. In some embodiments,the vaccine comprises a single bacterial, viral, protozoal, ortumor-related antigen in combination with one inventive compound. Insome embodiments, the vaccine comprises two or more bacterial, viral,protozoal, or tumor-related antigens in combination with a singleinventive compound. In some embodiments, the vaccine comprises a two ormore bacterial, viral, protozoal, or tumor-related antigens incombination with two or more inventive compounds. In some embodiments,the vaccine comprises a single bacterial, viral, protozoal, ortumor-related antigens in combination with two or more inventivecompounds.

In some embodiments, one or more antigens of provided vaccines arebacterial antigens. In certain embodiments, the bacterial antigens areantigens associated with a bacterium selected from the group consistingof Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis,Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp.,Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes,Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis,Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis,Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella pestis,Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni,Clostridium spp., Clostridium difficile, Mycobacterium spp.,Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borreliaburgdorferi, Leptospria spp., Hemophilus ducreyi, Corynebacteriumdiphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, hemophilus influenza, Escherichia coli, Shigella spp.,Erlichia spp., Rickettsia spp. and combinations thereof.

In certain embodiments, one or more antigens of provided vaccines areviral-associated antigens. In certain embodiments, the viral-associatedantigens are antigens associated with a virus selected from the groupconsisting of influenza viruses, parainfluenza viruses, mumps virus,adenoviruses, respiratory syncytial virus, Epstein-Barr virus,rhinoviruses, polioviruses, coxsackieviruses, echo viruses, rubeolavirus, rubella virus, varicell-zoster virus, herpes viruses, herpessimplex virus, parvoviruses, cytomegalovirus, hepatitis viruses, humanpapillomavirus, alphaviruses, flaviviruses, bunyaviruses, rabies virus,arenaviruses, filoviruses, HIV 1, HIV 2, HTLV-1, HTLV-II, FeLV, bovineLV, FeIV, canine distemper virus, canine contagious hepatitis virus,feline calicivirus, feline rhinotracheitis virus, TGE virus, foot andmouth disease virus, and combinations thereof.

In certain embodiments, one or more antigens of provided vaccines aretumor-associated antigens. In some embodiments, the tumor-associatedantigens are antigens selected from the group consisting of killed tumorcells and lysates thereof, MAGE-1, MAGE-3 and peptide fragments thereof;human chorionic gonadotropin and peptide fragments thereof;carcinoembryonic antigen and peptide fragments thereof, alphafetoprotein and peptide fragments thereof; pancreatic oncofetal antigenand peptide fragments thereof; MUC-1 and peptide fragments thereof, CA125, CA 15-3, CA 19-9, CA 549, CA 195 and peptide fragments thereof;prostate-specific antigens and peptide fragments thereof;prostate-specific membrane antigen and peptide fragments thereof;squamous cell carcinoma antigen and peptide fragments thereof; ovariancancer antigen and peptide fragments thereof; pancreas cancer associatedantigen and peptide fragments thereof; Her1/neu and peptide fragmentsthereof; gp-100 and peptide fragments thereof; mutant K-ras proteins andpeptide fragments thereof; mutant p53 and peptide fragments thereof;truncated epidermal growth factor receptor, chimeric proteinp210^(BCR-ABL), KH-1, N3, GM1, GM2, GD2, GD3, Gb3, Globo-H, STn, Tn,Lewis^(x), Lewis^(y), TF; and mixtures thereof.

In certain embodiments, an antigen is covalently bound to a compound offormula I, II, III, or IV. In some embodiments, an antigen is notcovalently bound to a compound of formula I, II, III, or IV.

One of ordinary skill in the art will appreciate that vaccines mayoptionally include a pharmaceutically acceptable excipient or carrier.Thus, according to another aspect, provided vaccines comprise one ormore antigens that are optionally conjugated to a pharmaceuticallyacceptable excipient or carrier. In some embodiments, said one or moreantigens are conjugated covalently to a pharmaceutically acceptableexcipient. In other embodiments, said one or more antigens arenon-covalently associated with a pharmaceutically acceptable excipient.

As described above, adjuvants may be used to increase the immuneresponse to an antigen. According to the invention, provided vaccinesmay be used invoke an immune response when administered to a subject. Incertain embodiments, an immune response to an antigen may be potentiatedby administering to a subject a provided vaccine in an effect amount topotentiate the immune response of said subject to said antigen.

As described above, provided compounds may be used in cancer vaccines asadjuvants in combination with tumor-associated antigens. In certainembodiments, said vaccines may be used in the treatment or prevention ofneoplasms. In certain embodiments, the neoplasm is a benign neoplasm. Inother embodiments, the neoplasm is a malignant neoplasm. Any cancer maybe treated using compounds of the invention with an antigen.

In certain embodiments, the malignancy is a hematological malignancy.Hematological malignancies are types of cancers that affect the blood,bone marrow, and/or lymph nodes. Examples of hematological malignanciesthat may be treated using compounds of formulae I, II, III, or IVinclude, but are not limited to, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin'slymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma (CTCL),peripheral T-cell lymphoma (PTCL), Mantle cell lymphoma, B-celllymphoma, acute lymphoblastic T cell leukemia (T-ALL), acutepromyelocytic leukemia, and multiple myeloma.

Other cancers besides hematological malignancies may also be treatedusing compounds of formulae I, II, III, or IV. In certain embodiments,the cancer is a solid tumor. Exemplary cancers that may be treated usingcompounds of formulae I, II, III, or IV include colon cancer, lungcancer, bone cancer, pancreatic cancer, stomach cancer, esophagealcancer, skin cancer, brain cancer, liver cancer, ovarian cancer,cervical cancer, uterine cancer, testicular cancer, prostate cancer,bladder cancer, kidney cancer, neuroendocrine cancer, breast cancer,gastric cancer, eye cancer, gallbladder cancer, laryngeal cancer, oralcancer, penile cancer, glandular tumors, rectal cancer, small intestinecancer, sarcoma, carcinoma, melanoma, urethral cancer, vaginal cancer,to name but a few.

In certain embodiments, compounds and pharmaceutical compositions of thepresent invention can be employed in combination therapies, that is, thecompounds and pharmaceutical compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, an inventive compoundmay be administered concurrently with another antiproliferative agent),or they may achieve different effects (e.g., control of any adverseeffects).

For example, other therapies or anticancer agents that may be used incombination with the inventive anticancer agents of the presentinvention include surgery, radiotherapy (γ-radiation, neutron beamradiotherapy, electron beam radiotherapy, proton therapy, brachytherapy,and systemic radioactive isotopes, to name a few), endocrine therapy,biologic response modifiers (interferons, interleukins, and tumornecrosis factor (TNF) to name a few), hyperthermia and cryotherapy,agents to attenuate any adverse effects (e.g., antiemetics), and otherapproved chemotherapeutic drugs, including, but not limited to,alkylating drugs (mechlorethamine, chlorambucil, Cyclophosphamide,Melphalan, Ifosfamide), antimetabolites (Methotrexate), purineantagonists and pyrimidine antagonists (6-Mercaptopurine,5-Fluorouracil, Cytarabile, Gemcitabine), spindle poisons (Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide,Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin),nitrosoureas (Carmustine, Lomustine), inorganic ions (Cisplatin,Carboplatin), enzymes (Asparaginase), and hormones (Tamoxifen,Leuprolide, Flutamide, and Megestrol), to name a few. Additionally, thepresent invention also encompasses the use of certain cytotoxic oranticancer agents currently in clinical trials and which may ultimatelybe approved by the FDA (including, but not limited to, epothilones andanalogues thereof and geldanamycins and analogues thereof). For a morecomprehensive discussion of updated cancer therapies see,www.nci.nih.gov, a list of the FDA approved oncology drugs atwww.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

In another aspect, the invention provides a method of treatinginfectious disease in a subject comprising administering to the subjecta therapeutically effective amount of a compound of formulae I, II, III,or IV. In some embodiments, the infection is bacterial. In someembodiments, the infection is viral. In some embodiments, the infectionis protozoal. In some embodiments, the subject is human.

Formulations

Inventive compounds may be combined with a pharmaceutically acceptableexcipient to form a pharmaceutical composition. In certain embodiments,the pharmaceutical composition includes a pharmaceutically acceptableamount of an inventive compound. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill vary depending upon the host being treated, and the particular modeof administration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, this amount will range from about 1% to about 99% of activeingredient, preferably from about 5% to about 70%, most preferably fromabout 10% to about 30%.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. In certain embodiments, a formulation ofthe present invention comprises an excipient selected from the groupconsisting of cyclodextrins, liposomes, micelle forming agents, e.g.,bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides;and a compound of the present invention. In certain embodiments, anaforementioned formulation renders orally bioavailable a compound of thepresent invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin, absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol, glycerol monostearate, and non-ionic surfactants;absorbents, such as kaolin and bentonite clay; lubricants, such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents. In the caseof capsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-shelled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In certain embodiments, a compound or pharmaceutical preparation isadministered orally. In other embodiments, the compound orpharmaceutical preparation is administered intravenously. Alternativerouts of administration include sublingual, intramuscular, andtransdermal administrations.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1% to 99.5% (morepreferably, 0.5% to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, a compound or pharmaceutical composition of theinvention is provided to a subject chronically. Chronic treatmentsinclude any form of repeated administration for an extended period oftime, such as repeated administrations for one or more months, between amonth and a year, one or more years, or longer. In many embodiments, achronic treatment involves administering a compound or pharmaceuticalcomposition of the invention repeatedly over the life of the subject.Preferred chronic treatments involve regular administrations, forexample one or more times a day, one or more times a week, or one ormore times a month. In general, a suitable dose such as a daily dose ofa compound of the invention will be that amount of the compound that isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Generally doses of the compounds of this invention for a patient, whenused for the indicated effects, will range from about 0.0001 to about100 mg per kg of body weight per day. Preferably the daily dosage willrange from 0.001 to 50 mg of compound per kg of body weight, and evenmore preferably from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

In some embodiments, provided adjuvant compounds are administered aspharmaceutical compositions or vaccines. In certain embodiments, theamount of adjuvant compound administered is 1-2000 μg. In certainembodiments, the amount of adjuvant compound administered is 1-1000 μg.In certain embodiments, the amount of adjuvant compound administered is1-500 μg. In certain embodiments, the amount of adjuvant compoundadministered is 1-250 μg. In certain embodiments, the amount of adjuvantcompound administered is 100-1000 μg. In certain embodiments, the amountof adjuvant compound administered is 100-500 μg. In certain embodiments,the amount of adjuvant compound administered is 100-200 μg. In certainembodiments, the amount of adjuvant compound administered is 250-500 μg.In certain embodiments, the amount of adjuvant compound administered is10-1000 μg. In certain embodiments, the amount of adjuvant compoundadministered is 500-1000 μg. In certain embodiments, the amount ofadjuvant compound administered is 50-250 μg. In certain embodiments, theamount of adjuvant compound administered is 50-500 μg.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, in certain embodiments the compound is administeredas a pharmaceutical formulation (composition) as described above.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The invention provides kits comprising pharmaceutical compositions of aninventive compound. In certain embodiments, such kits including thecombination of a compound of formulae I, II, III, or IV and an antigen.The agents may be packaged separately or together. The kit optionallyincludes instructions for prescribing the medication. In certainembodiments, the kit includes multiple doses of each agent. The kit mayinclude sufficient quantities of each component to treat a subject for aweek, two weeks, three weeks, four weeks, or multiple months. The kitmay include a full cycle of immunotherapy. In some embodiments, the kitincludes a vaccine comprising one or more bacterial, viral, protozoal,or tumor-associated antigens, and one or more provided compounds.

The entire contents of all references cited above and herein are herebyincorporated by reference.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

EXAMPLES Example 1

The synthesis of the hexasaccharide fragment within QS-7-Api (FIG. 1 )required initial preparation of the selectively protectedmonosaccharides 2-4, 6, and 8 (Scheme 7). While the xylo-, gluco- andapio-derived monosaccharides 2-4 (Scheme 7A) were obtained in multi-stepsequences by previously reported procedures and modifications thereof(Kim, Y. J.; Wang, P.; Navarro-Villalobos, M., Rohde, B. D.; Derryberry,J.; Gin, D. Y. J. Am. Chem. Soc. 2006, 128, 11906-11915; Nguyen, H. M.;Chen, Y. N.; Duron, S. G.; Gin, D. Y. J. Am. Chem. Soc. 2001, 123,8766-8772) the novel sugars 6 and 8 were prepared from rhamnopyranose 5and fucopyranoside 7, respectively. Silylation of theselectively-protected rhamnopyranose 5 (Scheme 7B) with TIPSOTf providedthe α-TIPS glycoside (96%), which subsequently underwentC4-O-debenzylation to furnish the rhamnopyranoside 6 (98%). Synthesis ofthe fucosyl residue within QS-7 commenced with selective C3-O-alkylationof the allyl fucopyranoside 7 (Scheme 7C) with PMBC1 (56%) via itstransient stannylene acetal. This allowed for sequential selectivesilylation of the equatorial C2-OH (97%) and acetylation of the axialC4-OH (>99%). Finally, oxidative removal of the PMB ether with DDQprovided the selectively-protected fucopyranoside 8 (86%).

Convergent assembly of the branched hexasaccharide (Scheme 8) involveddehydrative glycosylation (Ph₂SO.Tf₂O) (Garcia, B. A.; Gin, D. Y. J. Am.Chem. Soc. 2000, 122, 4269-4279) of fucopyranoside 8 with rhamnopyranose5 (84%). The resulting α-disaccharide 9 then underwent a series ofprotective group exchanges, including TBS removal (95%), a novelEt₂Zn/Pd(PPh₃)₄-mediated anomeric de-allylation (68%), (Chandrasekhar,S.; Reddy, C. R.; Rao, R. J. Tetrahedron 2001, 57, 3435-3438) andselective anomeric silylation (75%) to afford the disaccharide 10 as asuitable glycosyl acceptor. Its glycosyl donor coupling partner wasprepared by chemo- and stereoselective dehydrative glycosylation ofrhamnopyranoside 6 with xylopyranose 2 to afford the β-disaccharide11(75%), which directly underwent modified Helferich glycosylation(Roush, W. R.; Bennett, C. E. J. Am. Chem. Soc. 1999, 121, 3541-3542)with the apiose-derived donor 4 to afford trisaccharide 12 (86%). Theacetate esters in 12 were then exchanged for a benzylidene acetalprotective group (94%), followed by selective acid hydrolysis of therhamno-derived isopropylidene ketal to afford the corresponding vicinaldiol (71%). Selective alkylation of the resulting axial rhamno-C2-OHwith BnBr could then be accomplished (84%), allowing for Schmidtglycosylation (Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.1994, 50, 21-123) of the C3-OH with the glucosyl imidate 3 to afford thetetrasaccharide 13 (86%). Exchange of the benzoate ester for a TES ether(91%, 2 steps) and conversion of the anomeric TIPS group to itsα-trichloroacetimidate counterpart 14 (92%, 2 steps) secured a suitabledonor for glycosylation of disaccharide 10. This was accomplished bytreating the two components with TMSOTf to afford hexasaccharide 15(62%), whose fucosyl-TIPS-acetal was then transformed to theα-trichloroacetimidate 16 (84%, 2 steps).

Late stage construction of the full QS-7-Api skeleton involved theelaborately-protected triterpene-trisaccharide conjugate 18 (Scheme 9A),previously prepared from glucuronolactone 17 during the course of thesynthesis of QS-21 (Kim, et al., supra). This C28-carboxylic acidglycosyl acceptor 18 responded well to glycosylation withtrichloroacetimidate glycosyl donor 16 (BF₃.OEt₂) to afford fullyprotected QS-7-Api (71%), which underwent global deprotection undercarefully managed conditions (TFA; H₂, Pd—C). The resulting product(71%) was found to be identical to naturally derived QS-7-Api (1) (tracequantities of natural QS-7-Api (˜70% purity, NMR) were obtained fromexhaustive RP-HPLC purification of commercial Quil-A ($90/g).

This synthesis of 1 (Scheme 9A) from de novo construction of alloligosaccharide fragments confirms the structure of QS-7-Api andprovides significantly more dependable access to homogeneous samples of1 than isolation from natural sources. This notwithstanding, thesynthesis of 1 can be further augmented. Quil-A (19, Scheme 9B) is acommercially available semi-purified extract from Quillaja saponaria andcontains variable quantities of >50 distinct saponins (Vansetten,supra), many of which incorporate the triterpene-trisaccharidesubstructure within QS-7 (and QS-21). This monodesmoside saponin 20(Scheme 9B) can be isolated in semi-pure form via direct base hydrolysisof the Quil-A mixture (Higuchi et al., supra). Subsequentpoly(silylation) of 20 with excess TESOTf afforded the correspondingnonakis(triethylsilyl ether) (257 mg from 1.15 g of 19), whoseglucuronic acid functionality could be selectively derivatized to thebenzyl ester 21 (CbzCl, 68%). This triterpene-trisaccharide conjugate,obtained in only a 3-step protocol from Quil-A (19), was an effectivedonor in a C28-carboxylate glycosylation (800%) with hexasaccharide 16to provide, after global deprotection, QS-7-Api (1) (77%). The evolutionof the first synthesis of 1 to this semi-synthetic variant furnishescomplex QS-saponin adjuvants (and likely non-natural analogues) withmarkedly enhanced facility, enabling heretofore untapped opportunitiesfor novel adjuvant discovery in antitumor and antiviral vaccinedevelopment.

Experimental Procedures General Procedures.

Reactions were performed in flame-dried sealed-tubes or modified Schlenk(Kjeldahl shape) flasks fitted with a glass stopper under a positivepressure of argon, unless otherwise noted. Air- and moisture-sensitiveliquids and solutions were transferred via syringe. The appropriatecarbohydrate and sulfoxide reagents were dried via azeotropic removal ofwater with toluene. Molecular sieves were activated at 350° C. and werecrushed immediately prior to use, then flame-dried under vacuum. Organicsolutions were concentrated by rotary evaporation below 30° C. Flashcolumn chromatography was performed employing 230-400 mesh silica gel.Thin-layer chromatography was performed using glass plates pre-coated toa depth of 0.25 mm with 230-400 mesh silica gel impregnated with afluorescent indicator (254 nm).

Materials.

Lyophilized QS saponin Quil-A (batch L77-244) was obtained from BrenntagBiosector (Frederikssund, Denmark) via distribution by Accurate Chemicaland Scientific Corporation (Westbury, N.Y.). Dichloromethane,tetrahydrofuran, diethyl ether, hexane, toluene, and benzene werepurified by passage through two packed columns of neutral alumina underan argon atmosphere. Methanol was distilled from magnesium at 760 Torr.Trifluoromethanesulfonic anhydride was distilled from phosphoruspentoxide at 760 Torr. Boron trifluoride diethyl etherate and pyridinewere distilled from calcium hydride at 760 Torr. Dimethylformamide wasdried over 4 Å molecular sieves. All other chemicals were obtained fromcommercial vendors and were used without further purification unlessnoted otherwise.

Instrumentation.

Infrared (IR) spectra were obtained using a Perkin Elmer Spectrum BXspectrophotometer or a Bruker Tensor 27. Data are presented as thefrequency of absorption (cm⁻¹). Proton and carbon-13 nuclear magneticresonance (¹H NMR and ¹³C NMR) spectra were recorded on a Varian 400, aVarian 500, a Varian Inova 500, or a Bruker Avance III instrument,chemical shifts are expressed in parts per million (δ scale) downfieldfrom tetramethylsilane and are referenced to the residual protium in theNMR solvent (CHCl₃: δ 7.26 for ¹H NMR, δ 77.16 for ¹³C NMR). Data arepresented as follows: chemical shift, multiplicity (s=singlet, bs=broadsinglet, d=doublet, bd=broad doublet, t=triplet, q=quartet, m=multipletand/or multiple resonances), coupling constant in Hertz (Hz),integration, assignment. RP-HPLC purification and analyses were carriedout on a Waters 2545 binary gradient HPLC system equipped with a Waters2996 photodiode array detector, and absorbances were monitored at awavelength of 214 nm.

Preparation of the Hexasaccharide

O-Triisopropyl 4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranoside(S1). To a solution of rhamnopyranoside 5 (Nguyen, H. M.; Poole, J. L.;Gin, D. Y. Angew. Chem. Int. Ed. 2001, 40, 414-417) (6.00 g, 20.4 mmol,1.00 equiv) in dichloromethane (100 mL) at 0° C. was added 2,6-lutidine(8.30 mL, 71.4 mmol, 3.50 equiv) and triisopropylsilyltrifluoromethanesulfonate (9.30 mL, 34.7 mmol, 1.70 equiv). The reactionwas stirred at this temperature for 1 h and then at 23° C. for 3 h.Saturated aqueous NaHCO₃ (150 mL) was added, and the aqueous layer wasextracted with dichloromethane (3×150 mL). The combined organic phasewas washed with saturated aqueous NaCl (150 mL), dried (Na₂SO₄),filtered and concentrated. Silica gel chromatography (hexane/ethylacetate 20:1) afforded α-anomer S1 (8.8 g, 20 mmol, 96% yield) as acolorless liquid. ¹H NMR (CDCl₃, 500 MHz) δ 7.39-7.33 (m, 41H,aromatic), 7.30-7.26 (m, 11H, aromatic), 5.38 (s, 11H, H-1), 4.93 (d,J=10.2 Hz, 1H, PhCH ₂—), 4.65 (d, J=10.2 Hz, 1H, PhCH ₂—), 4.32 (dd,J=7.0, 5.6 Hz, 1H, H-3), 4.13 (d, J=5.6 Hz, 1H, H-2), 3.95 (qd, J=9.9,6.2 Hz, 1H, H-5), 3.25 (dd, J=9.9, 7.0 Hz, 1H, H-4), 1.53 (s, 3H, Me),1.40 (s, 3H, Me), 1.29 (d, J=6.3 Hz, 3H, Me), 1.18-1.08 (m, 21H,Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 138.51, 128.41, 128.17, 127.76,109.31, 91.80, 81.56, 78.80, 78.15, 73.25, 64.54, 28.23, 26.66, 17.89,17.81, 12.04; FTIR (neat film) 3032, 2941, 2896, 2868, 1463, 1382, 1370,1243, 1220, 1081, 1058, 1020, 995, 883 cm⁻¹.

O-Triisopropyl 2,3-di-O-isopropylidene-α-L-rhamnopyranoside (6). To asolution of Si (5.90 g, 13.1 mmol, 1.00 equiv) in methanol (100 mL) wasadded 10% (dry basis) palladium on carbon, wet, Degussa type E101 NE/W(1.4 g, 0.65 mmol, 0.050 equiv). The reaction mixture was vigorouslystirred under hydrogen pressure (110 psi) for 7.5 h and was thenfiltered through a Celite 545 plug, which was rinsed withdichloromethane. The filtrate and rinsings were concentrated, and theresidue was subjected to silica gel chromatography (hexane/ethyl acetate3:1) to afford 6 (4.6 g, 1.3 mmol, 98% yield) as a colorless oil. ¹H NMR(CDCl₃, 500 MHz) δ 5.34 (s, 1H, H-1), 4.16-4.10 (m, 2H, H-2 and H-3),3.91 (qd, J=8.8, 6.3 Hz, 1H, H-5), 3.42 (ddd, J=11.6, 6.8, 4.8 Hz, 1H,H-4), 2.33 (d, J=4.8 Hz, 1H, —OH), 1.53 (s, 3H, Me), 1.37 (s, 3H, Me),1.28 (d, J=6.3 Hz, 3H, Me), 1.18-1.05 (m, 21H, Si-i-Pr₃); ¹³C NMR (125MHz, CDCl₃) δ 109.5, 91.9, 78.2, 77.6, 74.4, 66.0, 28.0, 26.2, 17.8,17.68, 17.66, 11.9, FTIR (neat film) 3463 (br), 2942, 2868, 1464, 1383,1244, 1220, 1051, 1015, 883, 852, 807 cm⁻¹.

Allyl 3-O-methoxybenzyl-α-D-fucopyranoside (S2). Allyl fucoside 7 (111mg, 0.543 mmol, 1.00 equiv) and dibutyltin oxide (125 mg, 0.502 mmolequiv) in toluene (10 mL) were refluxed for 5 h in a Dean-Starkapparatus. After the reaction mixture cooled to 23° C., CsF (152 mg, 1.0mmol) was added, and the solvent was evaporated. Dimethylformamide (3.0mL) and p-methoxybenzyl chloride (0.136 mL, 1.0 mmol, 2.0 equiv) wereadded, and the reaction mixture was stirred at 23° C. for 48 h. Thesolvent was evaporated, and residue taken up in dichloromethane andfiltered. The filtrate and rinsings were concentrated, and silica gelchromatography (hexane/ethyl acetate 1:1) afforded S2 (98 mg, 0.30 mmol,56%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.32-7.29 (m, 2H),6.90-6.88 (m, 2H), 5.92 (m, 1H), 5.30 (dq, J=17.2, 1.6 Hz, 1H), 5.21(dq, J=10.4, 1.2 Hz, 1H), 4.93 (d, J=4.0 Hz, 1H), 4.68 (d, J=11.6 Hz,1H, PhCH ₂—), 4.64 (d, J=11.6 Hz, 1H, PhCH ₂—), 4.20 (ddt, J=12.8, 5.4,1.4 Hz, 1H), 4.05 (ddt, J=12.8, 6.2, 1.3 Hz, 1H), 4.00-3.89 (m, 2H),3.81 (s, 3H, OMe), 3.80 (m, 1H), 3.64 (dd, J=9.7, 3.2 Hz, 1H), 2.40 (s,1H), 2.11 (d, J=8.4 Hz, 1H), 1.30 (d, J=6.6 Hz, 3H); ¹³C NMR (125 MHz,CDCl₃) δ 159.5, 133.8, 130.0, 129.5, 117.7, 114.0, 97.7, 78.5, 71.8,69.5, 68.5, 68.3, 65.7, 55.3, 16.2. FTIR (neat film) 3462 (br), 3077,2979, 2907, 2838, 1612, 1514, 1249, 1088, 1037, 821 cm⁻¹.

O-Allyl 2-O-t-butyldimethylsilyl-3-O-methoxybenzyl-α-D-fucopyranoside(S3). Fucopyranoside S2 (205 mg, 0.632 mmol, 1.00 equiv),t-butyldimethylsilyl chloride (190 mg, 1.26 mmol, 1.99 equiv), imidazole(129 mg, 1.89 mmol, 3.00 equiv) and 4-(dimethylamino)-pyridine (6.2 mg,0.051 mmol, 0.080 equiv) were dissolved in dichloromethane (8.0 mL) andstirred at 23° C. for 27 h. The reaction mixture was directly purifiedby silica gel chromatography (hexane/ethyl acetate 7:3) to afford S3(270 mg, 0.62 mmol, 97% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.32-7.26 (m,2H), 6.92-6.85 (m, 2H), 5.94 (m, 1H), 5.34 (dq, J=17.4, 1.4 Hz, 1H),5.22 (dq, J=10.3, 1.3 Hz, 1H), 4.79 (d, J=3.8 Hz, 1H), 4.69 (d, J=11.3Hz, 1H, PhCH ₂—), 4.56 (d, J=11.3 Hz, 1H, PhCH ₂—), 4.19 (ddt, J=13.0,5.4, 1.3 Hz, 1H), 4.05 (ddt, J=13.2, 6.5, 1.2 Hz, 1H), 3.99 (m, 1H),3.95 (q, J=6.5 Hz, 1H), 3.82 (s, 3H, OMe), 3.76-3.70 (m, 2H), 2.50 (s,1H), 1.28 (d, J=6.6 Hz, 3H), 0.93 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H);¹³C NMR (125 MHz, CDCl₃) δ 159.31, 134.16, 130.43, 129.42, 117.73,113.83, 98.34, 78.27, 72.28, 70.11, 69.29, 68.48, 65.31, 55.21, 25.86,18.19, 16.17, −4.43, −4.69; FTIR (neat film) 3507 (br), 2953, 2930,2899, 2857, 1613, 1514, 1250, 1106, 1040, 875, 837, 778 cm⁻¹.

O-Allyl4-O-acetyl-2-O-t-butyldimethylsilyl-3-O-methoxybenzyl-α-D-fucopyranoside(S4). To fucopyranoside S3 (264 mg, 0.602 mmol, 1.00 equiv) and4-(dimethylamino)-pyridine (7.3 mg, 0.060 mmol, 0.10 equiv) indichloromethane (10 mL) was added triethylamine (0.25 mL, 1.8 mmol, 3.0equiv) and acetic anhydride (0.17 mL, 0.80 mmol, 3.0 equiv). Thereaction mixture was stirred at 23° C. for 22.5 h and then wasconcentrated and purified by silica gel chromatography (hexanes/ethylacetate 17:3) to afford S4 (288 mg, 0.599 mmol, >99% yield) as acolorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.24-7.21 (m, 2H), 6.85-6.82(m, 2H), 5.92 (m, 1H), 5.36 (dd, J=3.2, 1.0 Hz, 1H), 5.32 (dq, J=17.2,1.6 Hz, 1H), 5.21 (dq, J=10.4, 1.2 Hz, 1H), 4.81 (d, J=3.8 Hz, 1H), 4.60(d, J=10.6 Hz, 1H, PhCH ₂—), 4.40 (d, J=10.6 Hz, 1H, PhCH ₂—), 4.18(ddt, J=13.1, 5.2, 1.2 Hz, 1H), 4.12-4.02 (m, 2H), 3.97 (dd, J=9.9, 3.8Hz, 1H), 3.78 (s, 3H), 3.77 (m, 1H), 2.14 (s, 3H), 1.14 (d, J=6.5 Hz,3H), 0.89 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H); ¹³C NMR (125 MHz, CDCl₃)δ 170.84, 159.06, 134.07, 130.50, 129.58, 118.00, 113.56, 98.48, 76.12,71.61, 71.08, 69.56, 68.74, 64.85, 55.24, 25.91, 20.96, 18.30, 16.25,−4.35, −4.90; FTIR (neat film) 2983, 2954, 2930, 2902, 2857, 1742, 1614,1515, 1249, 1104, 1055. 1040, 1019, 837, 779, 735 cm⁻¹; HRMS (ESI) m/z:Calcd for C₂₅H₄₀O₇Si (M+Na⁺) 503.2441, found 503.2437.

O-Allyl 4-O-acetyl-2-O-t-butyldimethylsilyl-α-D-fucopyranoside (8). Tofucopyranoside S4 (100 mg, 0.208 mmol, 1.00 equiv) in dichloromethane (4mL) and H₂O (0.4 mL) at 0° C. was added2,3-dichloro-5,6-dicyano-1,4-quinone (71 mg, 0.31 mmol, 1.5 equiv).After stirring at 0° C. for 10 min and at 23° C. for 2.5 h, reactionmixture was filtered through Celite 435, concentrated, and purified bysilica gel chromatography (hexane/ethyl acetate 4:1) to afford 8 (65 mg,0.18 mmol, 86% yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 5.90(m, 1H), 5.32 (dq, J=17.1, 1.6 Hz, 1H), 5.25 (dd, J=3.5, 1.1 Hz, 1H),5.20 (dq, J=10.4, 1.3 Hz, 1H), 4.81 (d, J=3.7 Hz, 1H), 4.18 (ddt,J=13.1, 5.3, 1.5 Hz, 1H), 4.09 (qd, J=6.7, 1.0 Hz, 1H), 4.06 (dt,J=10.0, 3.2 Hz, 1H), 4.00 (qt, J=6.3, 1.2 Hz, 1H), 3.89 (dd, J=10.0, 3.7Hz, 1H), 2.16 (s, 3H), 2.05 (d, J=3.0 Hz, 1H), 1.13 (d, J=6.6 Hz, 3H),0.90 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃) δ171.12, 133.87, 117.79, 98.26, 73.27, 70.66, 68.80, 68.77, 65.03, 25.80,20.86, 18.22, 16.11, −4.51, −4.59; FTIR (neat film) 3503, 2927, 1737,1372, 1242, 1170, 1136, 1087, 1038, 939, 878, 839, 778 cm⁻¹; HRMS (ESI)m/z: Calcd for C₁₇H₃₂O₆Si (M+Na⁺) 383.1866, found 383.1864.

O-Allyl4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranose-(1→3)]-2-O-t-butyldimethylsilyl-α-D-fucopyranoside(9). Trifluoromethanesulfonic anhydride (0.39 mL, 2.3 mmol, 2.8 equiv)was added to a solution of rhamnopyranose 5 (245 mg, 0.832 mmol, 1.00equiv), phenyl sulfoxide (982 mg, 4.85 mmol, 5.83 equiv) and2,4,6-tri-t-butylpyridine (1.21 g, 4.89 mmol, 5.88 equiv) indichloromethane (25 mL) at −78° C. After the reaction was stirred at−78° C. for 30 min and at −45° C. for 1.5 h, a solution offucopyranoside 8 (150 mg, 0.416 mmol, 0.500 equiv) in dichloromethane(5.0 mL) was added via cannula. The reaction mixture was stirred at −45°C. for 1 h, at 0° C. for 1 h and at 23° C. for 14 h. Triethylamine (0.1mL) was added to the reaction mixture, which was concentrated andpurified by silica gel chromatography (hexanes/ethyl acetate 17:3) toafford 9 (222 mg, 0.349 mmol, 84% yield) as a colorless oil. ¹H NMR(CDCl₃, 400 MHz) δ 7.38-7.22 (m, 5H), 5.90 (m, 1H), 5.23-5.16 (m, 3H),4.88 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.70 (d, J=3.6 Hz, 1H), 4.63 (d,J=12.0 Hz, 1H, PhCH ₂—), 4.21-4.05 (m, 5H), 4.02-3.94 (m, 2H), 3.72 (dd,J=10.0, 6.5 Hz, 1H), 3.15 (dd, J=9.6, 6.0 Hz, 1H), 2.20 (s, 3H, Me),1.46 (s, 3H, Me), 1.32 (s, 3H, Me), 1.26 (d, J=6.0 Hz, 3H), 1.09 (d,J=6.8 Hz, 3H), 0.90 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H); ¹³C NMR (100MHz, CDCl₃) δ 170.6, 138.8, 134.1, 128.6, 128.4, 128.3, 128.2, 117.9,108.9, 99.7, 98.7, 81.0, 78.7, 76.3, 75.1, 73.9, 73.1, 70.2, 68.9,65.38, 65.35, 28.2, 26.4, 26.0, 21.1, 18.2, 17.8, 16.3, −4.2, −4.6; FTIR(neat film) 3066, 3033, 2985, 2934, 2857, 2905, 1747, 1455, 1382, 1373,1236, 1138, 1096, 1057, 1012, 937, 864, 777, 736, 698 cm⁻¹; HRMS (ESI)m/z: Calcd for C₃₃H₅₂O₁₀Si (M+NH₄ ⁺) 654.3674, found 654.3672.

O-Allyl4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranose-(1→3)]-α-D-fucopyranoside(S5). To a solution of disaccharide 9 (190 mg, 0.298 mmol, 1.00 equiv)in tetrahydrofuran (10 mL) at 0° C. was added tetrabutylammoniumfluoride solution (1.0 M in tetrahydrofuran, 0.33 mL, 0.33 mmol, 1.1equiv). After 15 min, the reaction mixture was warmed to 23° C. and wasstirred at this temperature for 4 h. Silica gel (1 g) was added, thesolvent was removed, and the reaction material was purified by silicagel chromatography (hexane/ethyl acetate 3:2) to afford S5 (148 mg,0.283 mmol, 95% yield). ¹H NMR (CDCl₃, 500 MHz) δ 7.37-7.24 (m, 5H),5.91 (m, 1H), 5.30 (m, 1H), 5.28 (s, 1H), 5.23 (m, 1H), 5.17 (dd, J=3.5,1.0 Hz, 1H), 4.94 (d, J=3.5 Hz, 1H), 4.86 (d, J=11.5 Hz, 1H, PhCH ₂—),4.64 (d, J=11.5 Hz, 1H, PhCH ₂—), 4.24-4.16 (m, 3H), 4.08-4.01 (m, 2H),3.99-3.88 (m, 2H), 3.72 (m, 1H), 3.18 (dd, J=9.5, 6.5 Hz, 1H), 2.13 (s,3H), 2.01 (d, J=10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H), 1.26 (d, J=6.5Hz, 3H), 1.11 (d, J=6.5 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 170.6,138.7, 133.7, 128.4, 128.2, 127.8, 118.4, 109.2, 99.5, 98.1, 80.9, 76.3,75.5, 73.3, 73.1, 69.4, 69.0, 65.8, 65.5, 28.2, 26.6, 21.0, 17.9, 16.4;FTIR (neat film) 3470 (br), 3032, 2985, 2936, 1744, 1454, 1381, 1237,1168, 1092, 933, 863, 816, 737, 698 cm⁻¹; HRMS (ESI) m/z: Calcd forC₂₇H₃₈O₁₀ (M+Na⁺) 545.2363, found 545.2355.

4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranose-(1→3)]-D-fucopyranose(S6). To disaccharide S5 (80 mg, 0.15 mmol, 1.0 equiv) and Pd(PPh₃)₄ (18mg, 0.015 mmol, 0.10 equiv) in diethyl ether (9.0 mL) was added Et₂Znsolution (1.0 M in hexane, 1.53 mL, 1.53 mmol, 10.0 equiv). The reactionmixture was stirred at 23° C. for 10 h and then another portion ofPd(PPh₃)₄ (18 mg, 0.015 mmol, 0.10 equiv) was added. After 11 h, thereaction was diluted with ethyl acetate, followed by the addition ofsaturated aqueous NaCl. The aqueous phase was extracted by ethyl acetate(2×50 mL). The combined organic phase was dried (MgSO₄), filtered,concentrated, and the residue was purified by silica gel chromatography(hexanes/ethyl acetate 1:4) to afford the hemiacetal S6 as a mixture ofanomers (50 mg, 0.10 mmol, 68% yield). Characteristic peaks: ¹H NMR (500MHz, CDCl₃) δ 7.37-7.25 (m, 5H), 4.87 (d, J=11.3 Hz, 1H, PhCH ₂—), 4.64(d, J=11.3 Hz, 1H, PhCH ₂—), 1.47 (s, 3H), 1.36 (s, 3H). The hemiacetalmixture was used immediately in the next silylation reaction.

O-Triisopropylsilyl4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranose-(1→3)]-β-D-fucopyranoside(10). To a solution of hemiacetal S6 (57 mg, 0.12 mmol, 1.0 equiv),imidazole (64 mg, 0.94 mmol, 8.0 equiv) and 4-(dimethylamino)-pyridine(3 mg, 0.02 mmol, 0.2 equiv) in dimethylformamide (0.5 mL) was treatedwith triisopropylsilylchloride (150 μL, 0.70 mmol, 5.9 equiv). Thereaction was stirred at 23° C. for 4 h, then directly purified by silicagel chromatography (hexanes/ethyl acetate 4:1) to afford 10 (56 mg,0.088 mmol, 75%) as a white powder. ¹H NMR (CDCl₃, 500 MHz) δ 7.38-7.25(m, 5H), 5.32 (s, 1H), 5.12 (dd, J=3.0, 1.3 Hz, 1H), 4.85 (d, J=12.0 Hz,1H, PhCH ₂—), 4.64 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.55 (d, J=7.5 Hz, 1H),4.18 (m, 2H), 3.82-3.64 (m, 4H), 3.18 (m, 1H), 2.18 (d, J=2.0 Hz, 1H),2.12 (s, 3H), 1.48 (s, 3H), 1.36 (s, 3H), 1.26 (d, J=6.5 Hz, 3H), 1.14(d, J=6.5 Hz, 3H), 1.10-1.02 (m, 21H, Si-i-Pr₃); ¹³C NMR (125 MHz,CDCl₃) δ 170.8, 138.7, 128.4, 128.2, 127.8, 109.2, 99.2, 97.9, 80.9,78.6, 76.7, 76.4, 74.2, 73.1, 72.7, 70.0, 65.5, 28.2, 26.6, 21.0, 18.05,18.00, 17.9, 16.5, 12.5; FTIR (neat film) 3496 (br), 3089, 3064, 3032,2938, 2866, 1744, 1455, 1381, 1237, 1076, 737 cm⁻¹; HRMS (ESI) m/z:Calcd for C₃₃H₅₄O₉Si (M+Na⁺) 645.3435, found 645.3421.

O-Triisopropylsilyl[2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]-2,3-di-O-isopropylidene-α-L-rhamnopyranoside(11). To a solution of xylopyranose 2 (140 mg, 0.424 mmol, 1.00 equiv),phenylsulfoxide (500 mg, 2.47 mmol, 5.83 equiv) and2,4,6-tri-1-butylpyridine (604 mg, 2.44 mmol, 5.78 equiv) indichloromethane (16 mL) at −78° C. was added trifluoromethanesulfonicanhydride (0.20 mL, 1.19 mmol, 2.80 equiv). After 15 min, a solution ofrhamnopyranoside 6 (305 mg, 0.846 mmol, 2.00 equiv) in dichloromethane(5 mL) was added via cannula. The reaction mixture was stirred at −78°C. for 15 min, at −45° C. for 30 min, at 0° C. for 30 min, at 23° C. for10 h, at 35° C. for 5 h, and finally at 23° C. for another 9 h. Thereaction mixture was diluted with dichloromethane (100 mL) and washedwith saturated aqueous NaHCO₃ (2×100 mL) and saturated aqueous NaCl(2×100 mL). The aqueous washings were extracted with dichloromethane(150 mL), and the combined organic phase was dried (MgSO₄), filtered,and concentrated to furnish a cream-colored amorphous solid. Silica gelchromatography (hexanes/ethyl acetate 7:3) afforded 11 (215 mg, 75%yield) as a white amorphous solid. ¹H NMR (CDCl₃, 500 MHz) δ 7.40-7.27(m, 10H), 5.37 (s, 1H), 4.94 (d, J=11.5 Hz, 1H, PhCH ₂—), 4.92 (d, J=7.2Hz, 1H), 4.75 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.66 (d, J=11.5 Hz, 1H, PhCH₂—), 4.64 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.22 (dd, J=7.0, 5.0 Hz, 1H),4.06 (dd, J=5.0, 0.5 Hz, 1H), 3.96 (dd, J=11.5, 5.5 Hz, 1H), 3.87 (m,1H), 3.72 (t, J=9.0 Hz, 1H), 3.64 (dd, J=10.0, 7.5 Hz, 1H), 3.53 (m,1H), 3.24 (d, J=10.0 Hz, 1H), 3.21 (t, J=9.5 Hz, 2H), 3.18 (d, J=9.0 Hz,1H), 1.51 (s, 3H), 1.36 (s, 3H), 1.26 (d, J=6.0 Hz, 3H), 1.18-1.05 (m,21H, Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.8, 138.7, 128.4, 128.2,127.8, 109.2, 99.2, 97.9, 80.9, 78.6, 76.7, 76.4, 74.2, 73.1, 72.7,70.0, 65.5, 28.2, 26.6, 21.0, 18.05, 18.00, 17.9, 16.5, 12.5; FTIR (neatfilm) 3483 (br), 3031, 2942, 2867, 1497, 1455, 1383, 1242, 1221, 1085,1018, 883, 809, 735, 697 cm⁻¹; HRMS (ESI) m/z: Calcd for C₃₇H₅₆O₉Si(M+Na⁺) 695.3591, found 695.3594.

O-Triisopropylsilyl{[2,3-di-O-acetyl-5-O-benzyl-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-2,3-di-O-isopropylidene-α-L-rhamnopyranoside(12). To a solution of 1,2,3-tri-O-acetyl-5-O-benzyl-β-D-apiofuranoside(4) (466 mg, 1.27 mmol, 1.89 equiv) and 11 (452 mg, 0.672 mmol, 1.00equiv) in dichloromethane (19 mL) at 0° C. was addedt-butyldimethylsilyl trifluoromethanesulfonate (7.7 μL, 0.034 mmol,0.050 equiv). After 25 min triethylamine (0.1 mL) was added. Thereaction mixture was concentrated, and the residue was purified bysilica gel chromatography (hexanes/ethyl acetate 4:1 to 3:2) to afford12 (567 mg, 0.579 mmol, 86% yield) as a colorless oil. R_(f)=0.58(hexanes/ethyl acetate 2:1); ¹H NMR (CDCl₃, 500 MHz) δ 7.37-7.23 (m,15H), 5.47 (s, 1H), 5.45 (s, 1H). 5.36 (s, 1H), 4.89 (d, J=1.5 Hz, 1H),4.86 (d, J=2.0 Hz, 1H), 4.63 (d, J=11.0 Hz, 1H), 4.56 (d, J=12.0 Hz, 1H,PhCH ₂—), 4.50 (d, J=11.5 Hz, 1H, PhCH ₂—), 4.42 (d, J=12.0 Hz, 1H, PhCH₂—), 4.39 (d, J=11.5 Hz, 1H, PhCH ₂—), 4.21 (d, J=10.5 Hz, 1H, PhCH ₂—),4.18 (dd, J=7.0, 5.5 Hz, 1H), 4.10 (d, J=10.5 Hz, 1H, PhCH ₂—), 4.07 (d,J=10.5 Hz, 1H), 4.03 (d, J=5.5 Hz, 1H), 3.90-3.82 (m, 3H), 3.76 (t,J=9.0 Hz, 1H), 3.62 (dd, J=10.0, 7.5 Hz, 1H), 3.30 (m, 1H), 3.24 (dd,J=9.5, 8.0 Hz, 1H), 3.14 (dd, J=12.0, 10.0 Hz, 1H), 2.04 (s, 3H), 1.97(s, 3H), 1.49 (s, 3H), 1.34 (s, 3H), 1.26 (d, J=6.5 Hz, 3H), 1.20-1.04(m, 21H, Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.3, 169.4, 138.7,138.3, 138.1, 128.7, 128.6, 128.4, 128.2, 128.09, 128.04, 127.99, 109.6,106.7, 101.8, 91.8, 85.9, 82.1, 78.9, 78.4, 78.3, 78.1, 77.5, 76.91,76.85, 74.5, 73.6, 73.5, 73.2, 69.7, 64.3, 64.0, 28.1, 26.8, 21.6, 20.8,18.03, 17.97, 17.94, 12.2; FTIR (neat film) 2943, 2868, 1747, 1455,1370, 1247, 1084, 883, 809 cm⁻¹; HRMS (ESI) m/z: Calcd for C₁₃H₇₄O₁₅Si(M+Na⁺) 1001.4695, found 1001.4730.

O-Triisopropylsilyl{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-2,3-di-O-isopropylidene-α-L-rhamnopyranoside(S7). Potassium carbonate (221 mg, 1.60 mmol, 6.99 equiv) was added to asolution of trisaccharide 12 (224 mg, 0.229 mmol, 1.00 equiv) inmethanol (10 mL) and water (1 mL). The reaction was stirred at 23° C.for 1 h. The reaction was diluted with saturated aqueous NH₄Cl solution(100 mL) and extracted with dichloromethane (3×100 mL). The combinedorganic phase were dried (Na₂SO₄) and concentrated in vacuo.

The residue was treated with α,α-dimethoxytoluene (25 mL) andp-toluenesulfonic acid monohydrate (22 mg, 0.11 mmol, 0.51 equiv) andstirred at 23° C. for 2 h. The reaction mixture was then diluted withdichloromethane (150 mL), and washed with saturated aqueous NaHCO₃ (100mL) and water (100 mL). The organic phase was dried (Na₂SO₄), filteredand concentrated in vacuo with heating to remove the excessα,α-dimethoxytoluene. The residue was purified by silica gelchromatography (hexanes/ethyl acetate 82:18) to give trisaccharide S7(211 mg, 0.215 mmol, 94% yield over two steps). ¹H NMR (CDCl₃, 400 MHz)δ 7.53-7.23 (m, 20H), 5.98 (s, 1H), 5.74 (s, 1H), 5.36 (s, 1H), 4.90 (d,J=7.6 Hz, 1H), 4.84 (d, J=11.0 Hz, 1H, PhCH ₂—), 4.69 (d, J=11.0 Hz, 1H,PhCH ₂—), 4.60 (d, 0.1=1.4 Hz, 2H), 4.53 (d, 0.1=11.6 Hz, 1H, PhCH ₂—),4.52 (s, 1H), 4.46 (d, J=11.7 Hz, 1H, PhCH ₂—), 4.19 (dd, J=5.4, 1.7 Hz,1H), 4.04 (dd, J=5.6, 0.7 Hz, 1H), 3.90-3.79 (m, 3H), 3.65 (s, 3H), 3.61(dd, J=7.3, 2.7 Hz, 1H), 3.27 (dd, J=7.8, 1.4 Hz, 1H), 3.16 (dd, J=10.1,1.4 Hz, 1H), 1.48 (s, 3H), 1.33 (s, 3H), 1.25 (d, J=6.5 Hz, 3H),1.20-1.02 (m, 21H, Si-i-Pr₃); ¹³C NMR (100 MHz, CDCl₃) δ 138.34, 138.28,138.20, 137.0, 129.9, 128.9, 128.7, 128.6, 128.54, 128.52, 128.04,127.96, 127.90, 127.7, 127.4, 109.6, 107.5, 106.6, 101.8, 91.9, 87.3,81.7, 78.4, 78.3, 78.1, 77.8, 76.8, 74.5, 73.8, 73.5, 73.2, 71.3, 64.3,64.0, 28.1, 26.′8, 18.04, 18.01, 17.95, 12.2; FTIR (neat film) 3090,3066, 3033, 2943, 2895, 2867, 1497, 1455, 1383, 1370, 1241, 1221, 1086,1055, 1019, 993, 883, 856, 808, 752, 735, 697 cm⁻¹; HRMS (ESI) m/z:Calcd for C₅₆H₇₄O₁₃Si (M+Na⁺) 1005.4796, found 1005.4838.

O-Triisopropylsilyl{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(S8). To a solution of trisaccharide S7 (133 mg, 0.135 mmol, 1.00 equiv)in a mixture of methanol (2.0 mL) and water (6 drops) was addedp-toluenesulfonic acid monohydrate (13 mg, 0.068 mmol, 0.51 equiv). Thereaction was stirred at 23° C. for 5 d and was then directly purified bysilica gel chromatography (hexanes/ethyl acetate 1:4 to 1:1) to affordS8 (90 mg, 0.095 mmol 71% yield) and starting material S7 (10 mg, 0.010mmol, 7.5% recovered). ¹H NMR (CDCl₃, 500 MHz) δ 7.52-7.50 (m, 2H),7.40-7.33 (m, 9H), 7.32-7.24 (m, 9H), 5.99 (s, 1H), 5.70 (s, 1H), 5.12(s, 1H), 4.83 (d, J=10.5 Hz, 1H, PhCH ₂—), 4.78 (d, J=10.5 Hz, 1H, PhCH₂—), 4.61 (d, 1H, J=2.0 Hz), 4.63 (d, J=12.5 Hz, 1H, PhCH ₂—), 4.59 (d,J=12.5 Hz, 1H, PhCH ₂—), 4.56-4.54 (m, 2H), 4.54 (d, J=11.5 Hz, 1H, PhCH₂—), 4.46 (d, J=11.5 Hz, 1H, PhCH ₂—), 3.99 (s, 2H), 3.90-3.78 (m, 5H),3.67 (m, 2H), 3.46 (t, J=9.0 Hz, 1H), 3.40-3.33 (m, 2H), 3.14 (dd,J=11.5, 10.5 Hz, 1H), 2.26 (d, J=3.0 Hz, 1H), 1.27 (d, J=6.5 Hz, 3H),1.16-1.04 (m, 21H, Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 138.2, 138.1,137.0, 136.9, 129.9, 128.95, 128.86, 128.7, 128.62, 128.55, 128.2,128.1, 128.0, 127.7, 127.4, 107.6, 106.7, 104.7, 93.9, 91.8, 87.3, 83.5,82.8, 78.6, 76.6, 76.1, 73.8, 73.5, 73.4, 72.9, 71.6, 71.0, 66.1, 64.4,17.97, 17.89, 17.7, 12.1; FTIR (neat film) 3468 (br), 3066, 3033, 2943,2867, 1455, 1386, 1093, 1053, 986, 882, 861, 734, 696 cm⁻¹; HRMS (ESI)m/z: Calcd for C₃₃H₇₀O₁₃Si (M+Na⁺) 965.4483, found 965.4470.

O-Triisopropylsilyl2-O-benzyl-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(S9). To a mixture of trisaccharide S8 (87 mg, 0.092 mmol, 1.0 equiv),dichloromethane (2.0 mL) and 20% aqueous NaOH (1.0 mL) was addedn-Bu₄NBr (6 mg, 0.02 mmol, 0.2 equiv) and benzyl bromide (0.11 mL, 0.92mmol, 10 equiv). After 20 h at 23° C., the reaction mixture wasconcentrated and the resulting residue was purified by silica gelchromatography (hexane/ethyl acetate 78:22) to afford S9 (80 mg, 84%yield). ¹H NMR (CDCl₃, 500 MHz) δ 7.52-7.50 (m, 2H), 7.40-7.24 (m, 23H),6.00 (s, 1H), 5.71 (s, 1H), 5.10 (s, 1H), 4.85 (d, J=11.0 Hz, 1H, PhCH₂—), 4.77 (d, J=11.0 Hz, 1H, PhCH ₂—), 4.71 (d, J=12.0 Hz, 1H, PhCH ₂—),4.70 (d, J=8.0 Hz, 1H), 4.66 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.63 (d,J=12.5 Hz, 1H, PhCH ₂—), 4.60 (d, J=12.5 Hz, 1H, PhCH ₂—), 4.54 (d,J=12.0 Hz, 1H, PhCH ₂—), 4.53 (s, 1H), 4.46 (d, J=12.0 Hz, 1H, PhCH ₂—),4.01-3.95 (m, 3H), 3.87-3.78 (m, 3H), 3.66 (s, 2H), 3.63 (dd, J=3.0, 1.5Hz, 1H), 3.57 (t, J=9.0 Hz, 1H), 3.38 (m, 1H), 3.28 (dd, J=9.5, 8.0 Hz,1H), 3.16 (t, J=11.5 Hz, 1H), 2.95 (d, J=6.5 Hz, 1H), 1.28 (d, J=6.0 Hz,3H), 1.08-0.97 (m, 21H, Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 138.2,138.1, 138.0, 137.2, 136.8, 129.7, 128.8, 128.51, 128.45, 128.43, 128.3,128.06, 128.04, 127.85, 127.82, 127.75, 127.5, 127.2, 107.4, 106.5,104.1, 92.3, 91.6, 87.1, 82.2, 82.1, 79.4, 78.1, 76.5, 75.2, 73.6,73.24, 73.17, 73.0, 71.6, 71.0, 66.4, 64.0, 17.76, 17.74, 17.69, 11.9;FTIR (neat film) 3477 (br), 3068, 3033, 2943, 1465 cm⁻¹; HRMS (ESI) m/z:Calcd for C₆₀H₇₆O₁₃Si (M+Na⁺) 1055.4953, found 1055.5006.

O-Triisopropylsilyl[2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)]-2-O-benzyl-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-1-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(13). To a solution of trisaccharide S9 (88 mg, 0.085 mmol, 1.0 equiv)and glucosyl imidate 3 (131 mg, 0.187 mmol, 2.20 equiv) in diethyl ether(5.0 mL) at −45° C. was added a solution of trimethylsilyltrifluoromethanesulphonate (2.3 μL, 0.013 mmol, 0.15 equiv) indichloromethane (115 μL). After 30 min at this temperature,triethylamine (0.15 mL) was added to the reaction mixture, which wasconcentrated and purified by silica gel chromatography (benzene/ethylacetate 19:1) to afford 13 (115 mg, 0.0733 mmol, 86% yield). ¹H NMR (500MHz, CDCl₃) δ 8.18 (d, J=7.5 Hz, 2H), 7.64-7.14 (m, 43H), 6.06 (s, 1H),5.91 (s, 1H), 5.34 (dd, J=9.5, 8.0 Hz, 1H), 4.98-4.92 (m, 3H), 4.86-4.81(m, 2H), 4.79 (s, 1H), 4.77 (d, J=11.0 Hz, 1H, PhCH ₂—), 4.75 (d, J=12.0Hz, 1H, PhCH ₂—), 4.70-4.58 (m, 6H), 4.47 (d, J=8.0 Hz, 1H), 4.42 (d,J=11.5 Hz, 1H, PhCH ₂—), 4.37 (d, J=11.5 Hz, 1H, PhCH ₂—), 4.32 (d,J=11.5 Hz, 1H), 4.20 (dd, J=9.0, 3.0 Hz, 1H), 4.15 (d, J=10.5 Hz, 1H,PhCH ₂—), 4.13 (d, J=10.5 Hz, 1H, PhCH ₂—), 3.88-3.70 (m, 7H), 3.66 (dd,J=11.5, 2.5 Hz, 1H), 3.59-3.53 (m, 2H), 3.44 (d, J=10.0 Hz, 1H),3.29-3.23 (m, 1H), 3.20 (dd, J=9.0, 8.0 Hz, 1H), 2.32 (m, 1H), 2.05 (t,J=11.0 Hz, 1H), 1.28 (d, J=6.0 Hz, 3H), 1.10-0.90 (m, 21H, Si-i-Pr₃);¹³C NMR (125 MHz, CDCl₃) δ 165.4, 139.2, 139.1, 138.71, 138.66, 138.64,138.2, 138.1, 136.9, 133.5, 130.2, 130.1, 129.9, 129.4, 128.94, 128.85,128.7, 128.60, 128.59, 128.57, 128.55, 128.45, 128.42, 128.1, 128.0,127.95, 127.93, 127.8, 127.7, 127.6, 127.54, 127.50, 127.4, 107.4,106.6, 102.7, 100.9, 94.0, 91.9, 87.4, 83.3, 81.8, 79.0, 78.3, 78.0,77.5, 76.7, 75.3, 74.9, 74.67, 74.64, 74.5, 73.89, 73.87, 73.84, 73.57,72.8, 71.4, 70.0, 67.3, 63.0, 18.10, 18.04, 18.02, 12.1; FTIR (neatfilm) 3030, 2938, 2862, 1734, 1452, 1264 cm⁻¹, HRMS (ESI) m/z: Calcd forC₉₄H₁₀₈O₁₉Si (M+Na⁺) 1569.7332, found 1569.7397.

O-Triisopropylsilyl2-O-benzyl-[3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)]-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(S10). To a solution of tetrasaccharide 13 (110 mg, 0.0701 mmol, 1.00equiv) in dichloromethane (15 mL) at −78° C. was addeddiisobutylaluminium hydride solution (1.0 M in hexane, 0.14 mL, 0.14mmol, 2.0 equiv). After 0.5 h, additional diisobutylaluminium hydridesolution (1.0 M in hexane, 0.20 mL, 0.20 mmol, 2.9 equiv) was added, and0.5 h later, the reaction was quenched with methanol at −78° C. Thereaction mixture was concentrated, and the residue was purified bysilica gel chromatography (hexanes/ethyl acetate 4:1) to afford S10 (95mg, 0.065 mmol, 92% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.50-7.42 (m, 2H),7.46-7.14 (m, 38H), 6.02 (s, 1H), 5.86 (s, 1H), 5.06 (d, J=8.0 Hz, 1H),4.97-4.92 (m, 2H), 4.88-4.80 (m, 4H), 4.72 (d, J=11.0 Hz, 1H), 4.68-4.60(m, 4H), 4.59-4.48 (m, 4H), 4.38 (d, J=12.0 Hz, 1H), 4.06-3.82 (m, 9H),3.70 (s, 4H), 3.62 (dd, J=11.0, 4.0 Hz, 1H), 3.54-3.40 (m, 4H),3.36-3.26 (m, 2H), 3.22 (d, J=7.5 Hz, 1H), 2.76 (m, 2H), 1.34 (d, J=6.0Hz, 3H), 1.10-0.90 (m, 21H, Si-i-Pr₃); ¹³C NMR (125 MHz, CDCl₃) δ 139.3,139.0, 138.48, 138.45, 138.2, 137.4, 136.8, 129.8, 129.4, 129.1, 128.9,128.71, 128.66, 128.59, 128.56, 128.48, 128.44, 128.37, 128.05, 127.99,127.7, 127.59, 127.57, 127.4, 107.8, 106.8, 105.0, 103.1, 93.7, 91.9,87.5, 85.1, 83.1, 82.8, 79.5, 78.2, 77.4, 77.2, 77.1, 76.1, 75.9, 75.1,74.7, 74.5, 73.9, 73.7, 73.6, 73.2, 71.3, 69.3, 67.2, 64.2, 18.2, 18.00,17.97, 12.1; FTIR (neat film) 3436 (br), 3059, 3032, 2930, 2863, 1497,1455, 1362, 1094, 883 cm⁻¹; HRMS (ESI) m/z: Calcd for C₈₇H₁₀₄O₁₈Si(M+NH₄ ⁺) 1482.7336, found 1482.7333.

O-Triisopropylsilyl2-O-benzyl-[3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-β-D-glucopyranosyl-(1→3)]-{(5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(S11). To a solution of tetrasaccharide S10 (90 mg, 0.061 mmol, 1.0equiv) in dichloromethane (10 mL) at 0° C. was added 2,6-lutidine (0.21mL, 1.8 mmol, 26 equiv) and triethylsilyl trifluoromethanesulfonate(0.21 mL, 0.92 mmol, 15 equiv). The reaction mixture was stirred at thistemperature for 30 min and at 23° C. for 8 h. The reaction mixture wasconcentrated, and the residue was purified by silica gel chromatography(hexanes/ethyl acetate 23:2) to afford S11 (96 mg, 0.061 mmol, 99%yield). ¹H NMR (500 MHz, CDCl₃) δ 7.50-7.44 (m, 2H), 7.38-7.02 (m, 38H),5.96 (s, 1H), 5.89 (s, 1H), 5.03 (d, J=8.0 Hz, 1H), 4.95 (d, J=11.5 Hz,1H, PhCH ₂—), 4.89-4.82 (m, 3H), 4.80 (d, J=12.5 Hz, 1H, PhCH ₂—), 4.75(d, J=11.1 Hz, 1H, PhCH ₂—), 4.71 (d, J=11.2 Hz, 1H, PhCH ₂—), 4.64 (d,J=7.5 Hz, 1H), 4.63-4.48 (m, 7H), 4.46 (d, 0.1=11.3 Hz, PhCH ₂ —, 1H),4.39 (d, J=12.5 Hz, 1H, PhCH ₂—), 4.20 (dd, 2H, 1H, J=9.0, 3.5 Hz), 4.01(t, J=9.5 Hz, 1H), 3.98-3.78 (m, 5H), 3.65-3.54 (m, 4H), 3.50-3.38 (m,3H), 3.36-3.24 (m, 3H), 3.22 (dd, J=9.5, 8.0 Hz, 1H), 2.11 (d, J=9.5 Hz,1H), 1.31 (d, J=6.0 Hz, 3H), 1.06-0.89 (m, 29H), 0.72 (m, 6H), 0.56 (m,1H); ¹³C NMR (125 MHz, CDCl₃) δ 139.4, 139.3, 139.0, 138.8, 138.3,138.2, 137.0, 129.8, 128.9, 128.7, 128.6, 128.50, 128.45, 128.43, 128.4,128.3, 128.2, 127.93, 127.90, 127.8, 127.74, 127.70, 127.66, 127.6,127.5, 127.3, 127.2, 107.5, 106.7, 102.7, 102.1, 93.7, 91.9, 87.4, 85.0,82.9, 79.3, 78.2, 77.5, 77.35, 77.32, 76.1, 75.7, 75.2, 74.6, 74.5,74.0, 73.80, 73.78, 73.4, 73.0, 71.3, 69.0, 66.9, 64.1, 18.1, 18.0,12.1, 7.5, 6.9, 6.1, 5.8; FTIR (neat film) 3065, 3032, 2943, 2871, 1497,1455, 1364, 1178, 1148, 1094, 1028, 884, 735, 697 cm⁻¹; HRMS (ESI) m/z:Calcd for C₉₃H₁₁₈O₁₈Si₂ (M+Na⁺) 1601.7754, found 1601.7816.

2-O-benzyl-[3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-β-D-glucopyranosyl-(1→3)]-([5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)])-L-rhamnopyranose(S12). To a solution of tetrasaccharide SI 1 (96 mg, 0.061 mmol, 1.0equiv) in tetrahydrofuran (6 mL) at 0° C. was added tetrabutylammoniumfluoride solution (1.0 M in tetrahydrofuran, 63 μL, 0.063 mmol, 1.0equiv). After 3 min, silica gel (120 mg) was added to the reactionmixture, which was concentrated and purified by silica gelchromatography (hexanes/ethyl acetate 11:9) to afford hemiacetal S12 (88mg, quantitative yield). ¹H NMR (500 MHz, CDCl₃) δ 7.49-7.45 (m, 2H),7.36-7.09 (m, 38H), 5.94 (s, 1H), 5.80 (s, 1H), 5.01 (dd, J=3.0, 2.8 Hz,1H), 4.98-4.90 (m, 2H), 4.88-4.76 (m, 3H), 4.76-4.70 (m, 2H), 4.68-4.60(m, 2H), 4.60-4.53 (m, 2H), 4.53-4.44 (m, 6H), 4.22 (dd, J=8.5, 3.5 Hz,1H), 4.00-3.76 (m, 7H), 3.74 (dd, J=3.0, 2.0 Hz, 1H), 3.64-3.56 (m, 2H),3.54-3.20 (m, 9H), 2.68 (m, 1H), 2.37 (d, J=3.5 Hz, 1H), 1.32 (d, J=6.5Hz, 3H), 0.98 (m, 9H), 0.68-0.52 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ139.3, 138.9, 138.8, 138.7, 138.1, 137.4, 137.0, 128.8, 128.6, 128.54,128.50, 128.45, 128.43, 128.40, 128.37, 128.3, 128.2, 127.9, 127.8,127.71, 127.68, 127.64, 127.60, 127.57, 127.54, 127.4, 127.35, 127.30,127.2, 107.4, 106.6, 102.0, 91.80, 91.78, 87.3, 82.4, 78.7, 78.4, 76.5,75.9, 75.6, 74.9, 74.8, 74.6, 73.7, 73.5, 73.4, 72.9, 71.3, 69.1, 67.4,18.2, 18.1, 17.9, 13.1, 12.5, 7.4, 5.7, 5.6; FTIR (neat film) 3401 (br),3032, 2938, 2874, 1454, 1364, 1095, 1066, 734, 696 cm⁻¹, HRMS (ESI) m/z:Calcd for C₈₄H₉₈O₁₈Si (M+Na⁺) 1445.6420, found 1445.6449.

O-Trichloroacetimidoyl2-O-benzyl-[3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-β-D-glucopyranosyl-(1→3)]-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside(14). To a solution of hemiacetal S12 (64 mg, 0.045 mmol, 1.0 equiv) indichloromethane (10 mL) at 0° C. was added trichloroacetonitrile (0.9mL, 0.9 mmol, 200 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (62 μL,0.045 mmol, 10 equiv). After 3 h at 0° C., triethylamine (100 μL) wasadded. The reaction mixture was concentrated, and the residue waspurified by silica gel chromatography (hexanes/ethyl acetate 7:3) toafford 14 (65 mg, 92% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.52 (s, 1H),7.53-7.49 (m, 2H), 7.41-7.09 (m, 38H), 6.17 (d, J=2.0 Hz, 1H), 6.00 (s,1H), 5.90 (s, 1H), 5.04 (d, J=7.8 Hz, 1H), 4.97 (d, J=11.8 Hz, 1H, PhCH₂—), 4.89 (d, J=11.8 Hz, 1H, PhCH ₂—), 4.85 (d, J=12.2 Hz, 1H, PhCH ₂—),4.84 (d, J=11.1 Hz, 1H, PhCH ₂—), 4.78 (d, J=11.1 Hz, 1H, PhCH ₂—), 4.74(d, J=11.3 Hz, 1H, PhCH ₂—), 4.71 (d, J=11.3 Hz, 1H, PhCH ₂—), 4.65-4.45(m, 9H), 4.39 (d, J=12.4 Hz, 1H, PhCH ₂—), 4.05-3.87 (m, 6H), 3.67 (d,J=10.5 Hz, 1H, PhCH ₂—), 3.64 (d, J=10.5 Hz, 1H, PhCH ₂—), 3.57 (t,J=9.4 Hz, 1H), 3.50-3.40 (m, 3H), 3.40-3.22 (m, 4H), 2.20 (d, J=9.3 Hz,1H), 1.41 (d, J=6.2 Hz, 3H), 1.00-0.92 (m, 9H), 0.80-0.68 (m, 6H); ¹³CNMR (125 MHz, CDCl₃) δ 160.1, 139.1, 138.9, 138.5, 138.35, 138.30,138.0, 137.9, 136.8, 129.6, 128.7, 128.4, 128.3, 128.24, 128.23, 128.13,128.09, 128.0, 127.8, 127.75, 127.71, 127.6, 127.50, 127.48, 127.46,127.34, 127.32, 127.29, 127.2, 127.1, 127.0, 107.3, 106.4, 102.4, 101.7,96.4, 91.7, 91.4, 87.2, 84.8, 82.6, 78.0, 77.0, 76.9, 76.5, 76.1, 75.6,75.4, 75.0, 74.31, 74.26, 73.6, 73.49, 73.46, 73.2, 72.8, 71.1, 70.1,68.5, 18.0, 7.2, 5.5; FTIR (neat film) 3335, 3063, 3030, 2935, 2876,1673, 1454, 1363, 1176, 1149, 1095, 1063, 1028, 796, 734, 697 cm⁻¹.

O-Triisopropylsilyl(2-O-benzyl-[3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-β-D-glucopyranosyl-(1→3)]-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside-[1→2])-4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-αL-rhamnopyranose-(1→3)]-β-D-fucopyranoside (15). Dichloromethane (1.5mL) was added to disaccharide 10 (18 mg, 0.028 mmol, 2.8 equiv), imidate14 (16 mg, 0.010 mmol, 1.0 equiv), and 4 Å molecular sieves (40 mg), andthe resulting mixture was stirred for 30 min at 23° C. and was thencooled to −15° C. A solution of trimethylsilyltrifluoromethanesulphonate (0.145 μL, 0.000799 mmol, 0.0783 equiv) indichloromethane (20 μL) was added. After 40 min triethylamine (60 μL)was added, and the reaction mixture was filtered, concentrated, andpurified by silica gel chromatography (benzene/ethyl acetate 97:3) toafford 15 (13 mg, 62% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.46-7.42 (m,2H), 7.37-7.07 (m, 43H), 5.90 (s, 1H), 5.72 (s, 1H), 5.26 (d, J=2.0 Hz,1H), 5.09 (s, 1H), 4.92-4.76 (m, 5H), 4.71 (d, J=11.5 Hz, 1H, PhCH ₂—),4.65 (d, J=12.0 Hz, 1H, PhCH ₂—), 4.62-4.36 (m, 11H), 4.18 (m, 1H), 4.08(m, 2H), 3.96-3.82 (m, 5H), 3.73 (m, 1H), 3.67 (dd, J=10.0, 6.5 Hz, 1H),3.64-3.48 (m, 5H), 3.42-3.34 (m, 2H), 3.34-3.26 (m, 3H), 3.24-3.16 (m,2H), 3.12 (dd, J=9.5, 7.0 Hz, 1H), 2.11 (s, 3H), 1.34-1.24 (m, 9H),1.18-1.02 (m, 21H), 0.84-0.74 (m, 9H), 0.64-0.54 (m, 6H); ¹³C NMR (125MHz, CDCl₃) δ 171.0, 139.2, 138.7, 138.2, 136.9, 129.8, 129.4, 128.8,128.65, 128.55, 128.43, 128.38, 128.2, 128.04, 128.00, 127.84, 127.78,127.75, 127.66, 127.61, 127.4, 127.3, 127.1, 109.3, 107.3, 106.4, 97.0,91.7, 87.2, 80.4, 79.0, 78.7, 78.5, 77.1, 76.8, 76.4, 75.56, 75.4, 74.6,74.0, 73.7, 73.4, 73.0, 72.8, 71.3, 65.7, 63.9, 41.6, 29.9, 28.0, 26.2,21.1, 18.8, 18.4, 18.3, 17.8, 16.5, 12.87, 7.3, 5.5; FTIR (neat film)2930, 2868, 1744, 1068 cm⁻¹; LRMS (MALDI) m/z: Calcd for C₁₁₇H₁₅₀O₂₇Si₂(M+Na) 2065.98, found 2066.60.

O-Trichloroacetimidoyl(2-O-benzyl-[3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-β-D-glucopyranosyl-(1→3)]-{[5-O-benzyl-2,3-di-O-benzylidene-β-D-apiofuranosyl-(1→3)]-2,4-di-O-benzyl-β-D-xylopyranosyl-(1→4)]}-α-L-rhamnopyranoside-[1→2])-4-O-acetyl-[4-O-benzyl-2,3-di-O-isopropylidene-α-L-rhamnopyranose-(1→3)]-α-D-fucopyranoside17. To a solution of 15 (10.0 mg, 0.00489 mmol, 1.00 equiv) intetrahydrofuran (1.50 mL) at 0° C. was added tetrabutylammonium fluoridesolution (0.0245 M in tetrahydrofuran, 0.20 mL, 0.0049 mmol, 1.0 equiv).After 50 min, the solvent was removed in vacuo at 0° C. to give a paleyellow oil that was immediately subjected to further reaction.R_(f)=0.42 (benzene/ethyl acetate 9:1). The hemiacetal residue was driedby azeotropic removal of water with toluene (3×1 mL) and was thendissolved in dichloromethane (4.0 mL) and cooled to 0° C.Trichloroacetonitrile (74 μL, 0.74 mmol, 150 equiv) and1,8-diazabicyclo[5.4.0]undec-7-ene (2.7 μL, 0.020 mmol, 4.0 equiv) wereadded, and the solution was stirred at 0° C. for 13.5 h and 23° C. for 2h. The solution was then concentrated and purified by silica gelchromatography (benzene/ethyl acetate 99:1 to 9:1) to afford 16 (8.3 mg,0.0041 mmol, 84% yield). R_(f)=0.53 (benzene/ethyl acetate 9:1); ¹H NMR(500 MHz, CDCl₃) δ 8.54 (s, 1H, C═NH), 7.49-6.95 (m, 45H), 6.41 (d,J=3.5 Hz, 1H), 5.90 (s, 1H), 5.68 (s, 1H), 5.28-5.20 (m, 2H), 5.04 (d,J=11.9 Hz, 1H), 4.92 (d, J=11.9 Hz, 1H), 4.87-4.64 (m, 3H), 4.84 (d,J=11.7 Hz, 1H), 4.83 (d, J=11.7 Hz, 1H), 4.68 (d, J=10.9 Hz, 1H),4.64-4.39 (m, 11H), 4.32 (s, 1H), 4.28-4.19 (m, 3H), 4.19-4.08 (m, 3H),3.91-3.78 (m, 4H), 3.78-3.61 (m, 3H), 3.57-3.33 (m, 6H), 3.30 (m, 1H),3.23-3.09 (m, 3H), 2.16 (s, 3H, —C(O)CH ₃), 1.34 (s, 3H), 1.32-1.23 (m,10H), 1.14 (d, J=6.5 Hz, 3H), 1.12 (s, 3H), 0.85 (t, J=7.8 Hz, 9H),0.66-0.56 (m, 6H); FTIR (neat film) 2932, 2875, 1746, 1673, 1497, 1454,1367, 1229, 1098, 1072, 990, 735, 698 cm⁻¹; LRMS (MALDI) m/z: Calcd forC₁₁₀H₁₃₀Cl₃NO₂₇NCl₃SiNa (M+Na) 2055.64, found 2055.73.

Final Assembly of Synthetic Qs-7-Api

Fully-protected synthetic QS-7-Api (S13). A solution of borontrifluoride diethyl etherate (0.40 μL, 0.0032 mmol, 1.0 equiv) indichloromethane (20 μL) was injected to a solution of the imidate 16(6.4 mg, 0.0031 mmol, 1.0 equiv), 18 (9.1 mg, 0.0047 mmol, 1.5 equiv)and 4 Å molecular sieves (20 mg) in dichloromethane (2.0 mL) at −78° C.The reaction temperature was allowed to warm to 23° C. slowly, andtriethylamine (20 μL) was added after 16.5 h. The reaction wasconcentrated and purified by silica gel chromatography (hexanes/ethylacetate 4:1) to afford S13 (8.5 mg, 0.0022 mmol, 71% yield) and startingmaterial 18 (3.0 mg, 0.0016 mmol, 33% recovered). R_(f)=0.67(benzene/ethyl acetate 7:3); characteristic resonances from ¹H NMR (500MHz, CDCl₃) δ 9.41 (s, 1H, —CHO), 5.94 (s, 1H), 5.83 (s, 1H), 5.30 (d,J=7.3 Hz, 1H), 5.23 (m, 1H), 5.23 (d, J=12.4 Hz, 1H), 5.13 (d, J=12.4Hz, 1H), 5.08 (s, 1H), 5.01 (in, 1H), 4.31 (t, J=6.6 Hz, 1H), 4.22 (m,1H), 4.17 (d, J=7.1 Hz, 1H), 4.12 (q, J=7.1 Hz, 1H), 2.96 (m, 1H), 2.83(m, 1H), 2.72 (m, 1H), 2.50 (m, 1H), 2.09 (s, 31H, —C(O)CH ₃), 1.39 (s,3H, Me), 1.30 (s, 3H, Me), 1.12 (s, 3H, Me); see proton NMR below; MS(MALDI) m/z: Calcd for C₂₂₂H₂₈₄O₄₆Si₄Na (M+Na) 3821 (±2), found 3820(±2).

Synthetic QS-7-Api (1). Two solutions of fully-protected QS-7-Api S13(1.3 mg, 0.00034 mmol, 1.0 equiv) in dichloromethane (0.20 mL) were eachtransferred to a 10-mL round bottom flask and cooled to 0° C. Apre-cooled (0° C.) solution of trifluoroacetic acid (1.0 mL, TFA/water4:1) was added to each flask. After vigorous stirring for 60 min, thereaction mixtures were concentrated in vacuo for 140 min at 0° C. togive a white solid residue.

To both flasks were added tetrahydrofuran (1.0 mL), ethanol (2.0 mL),and 10% (dry basis) palladium on carbon, wet, Degussa type E101 NE/W(2.0 mg, 0.00094 mmol, 2.8 equiv). The two runs were stirred underhydrogen pressure (50 psi) for 44 h, and then the suspensions werecombined and filtered through a 0.45 pm polyvinylidene fluoride filterdisk, which was then washed with methanol (5 mL). The filtrate andrinsing were concentrated and purified by RP-HPLC on an XBridge PrepBEH300 C18 column (5 μm, 10×250 mm) using a linear gradient of 32→37%acetonitrile (0.05% TFA) in water (0.05% TFA) over 30 min at a flow rateof 5 mL/min. The fraction containing the major peak (t_(R)=14.85 min)was collected and lyophilized to dryness to afford synthetic QS-7-Api(1) (0.9 mg, 0.0005 mmol, 70% yield) as a white solid. ¹H NMR (500 MHz,7:3 D₂O:CD₃CN) δ 9.36 (s, 1H, —CHO), 5.39 (d, J=8.0 Hz, 1H), 5.34 (m,1H, R₂C═CHR), 5.22 (d, J=3.1 Hz, 1H), 5.12 (d, J=2.9 Hz, 1H), 5.03 (s,1H), 4.86 (s, 1H), 4.67 (d, J=7.7 Hz, 1H), 4.63 (d, J=7.8 Hz, 1H), 4.55(d, J=7.7 Hz, 1H), 4.50 (d, J=7.7 Hz, 1H), 4.33 (m, 1H), 4.05 (d, J=10.1Hz, 1H), 4.02 (m, 1H), 3.99-3.94 (m, 2H), 3.92-3.57 (m, 22H), 3.55-3.45(m, 7H), 4.33 (m, 1H), 3.42-3.15 (m, 12H), 2.86 (dd, J=14.5, 3.6 Hz,1H), 2.14 (s, 31H, —C(O)CH ₃), 1.89-1.30 (m, 14H), 1.28 (s, 3H, Me),1.22 (d, J=6.2 Hz, 3H, Me), 1.15 (d, J=6.1 Hz, 3H, Me), 1.08 (s, 3H,Me), 1.05 (m, 1H), 1.02 (m, 1H), 0.99 (d, J=6.3 Hz, 3H, Me), 0.93 (s,3H, Me), 0.89 (s, 3H, Me), 0.84 (s, 3H, Me), 0.71 (s, 3H, Me).

Natural QS-7-Api (1). Brenntag Quil-A (205 mg, batch L77-244) wasfractionated by RP-HPLC on an XBridge Prep C18 OBD column (5 μm, 19×150mm) using a linear gradient of 30→40% acetonitrile (0.05% TFA) in water(0.05% TFA) over 30 min at a flow rate of 15 mL/min. Crude naturalQS-7-Api (1) (t_(R)=15.25 min) was collected and further purified on anXBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a linear gradientof 32→37% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30 min at aflow rate of 5 mL/min. The fraction containing QS-7-Api (1) (t_(R)=14.85min) was collected and lyophilized to dryness to afford natural QS-7-Api(1) (2.0 mg) as a white solid (˜70% pure by ¹H NMR).

Preparation of the Semisynthetic Triterpene-Trisaccharide

O-(16-O-triethylsilyl-quillaic acid)4-O-triethylsilyl-[(2,3,4-tri-O-triethylsilyl-β-D-xylopyranosyl)-(1→3)]-[(2,3,4,6-tetra-O-triethylsilyl-β-D-galactopyranosyl-(1→2)]-β-D-glucuronoside(S14) and O-(16-O-triethylsilyl-quillaic acid)4-O-triethylsilyl-1(2,3,4-tri-O-triethylsilyl-α-L-rhamnopyranosyl)-(1→3)]-[(2,3,4,6-tetra-O-triethylsilyl-β-D-galactopyranosyl-(1→3)]-β-glucuronoside(S16). A mixture of Brenntag Quil A (1.15 g, batch L77-244) andpotassium hydroxide (0.97 g) in ethanol (25 mL) and water (25 mL) washeated at 80° C. for 7.25 h, then cooled to 0° C. and neutralized with 1N aqueous NaOH. The reaction mixture was concentrated to one-half volumeand purified by silica gel chromatography(chloroform/methanol/water/acetic acid 15:9:2:1). The major product spotby TLC was isolated, concentrated, and dried by azeotropic removal ofsolvents with toluene (2×20 mL) and lyophilization from water (4×30 mL)to afford a mixture of prosapogenins as a light tan foam (0.576 g).R_(f)=0.44 (chloroform/methanol/water/acetic acid 15:9:2:1);characteristic resonances from ¹H NMR: (500 MHz, CD₃OD) δ 9.44 (s, 1H,—CHO), 5.33 (m, 1H, R₂C═CHR), 4.80 (d, J=7.4 Hz, 1H), 4.63 (d, J=7.7 Hz,1H), 4.43 (s, 1H), 4.37 (d, J=6.9 Hz, 1H); ¹H NMR (500 MHz, 7:3D₂O:CD₃CN) δ 9.36 (s, 1H, —CHO), 5.29 (m, 1H, R₂C═CHR), 4.68 (d, J=7.7Hz, 1H), 4.59 (d, J=8.2 Hz, 1H).

Pyridine (8 mL) was added to the prosapogenin (0.560 g), and thesuspension was concentrated. Additional pyridine (8.6 mL) was added,followed by triethylsilyl trifluoromethanesulfonate (1.98 mL, 8.76mmol). Further triethylsilyl trifluoromethanesulfonate was added to thereaction after 66 h (0.33 mL, 1.5 mmol), 89 h (66 μL, 0.29 mmol), and112 h (0.13 mL, 0.54 mmol). After 5 d, the reaction mixture wasconcentrated and passed through a plug of silica gel (hexane/ethylacetate 4:1 to 7:3) to give a light yellow oil that was then dissolvedin methanol (10 mL) and tetrahydrofuran (10 mL). The solution wasstirred for 3.5 d, and then it was concentrated and purified by silicagel chromatography (hexane/ethyl acetate 4:1 to 7:3) to afford diacidS14 (0.257 g) and diacid S16 (0.095 g) as white solids.

S14 R_(f)=0.39 (benzene/ethyl acetate 4:1); ¹H NMR (500 MHz, CDCl₃) δ9.63 (s, 1H, —CHO), 5.33 (m, 1H, R₂C═CHR), 4.54 (m, 1H), 4.50 (d, J=7.3Hz, 1H), 4.44 (d, J=5.9 Hz, 1H), 4.39 (d, J=7.4 Hz, 1H), 3.95-3.87 (m,4H), 3.83-3.78 (m, 2H), 3.74 (t, J=9.0 Hz, 1H), 3.65-3.58 (m, 3H), 3.48(m, 1H), 3.43-3.31 (m, 3H), 3.25 (t, J=7.9 Hz, 1H), 3.12 (t, J=10.8 Hz,1H), 2.95 (dd, J=13.9, 3.7 Hz, 1H), 2.20 (t, J=13.3 Hz, 1H), 1.91-1.04(m, 23H), 1.34 (s, 3H, Me), 1.23 (s, 3H, Me), 1.04-0.91 (m, 86H), 0.89(s, 3H, Me), 0.80-0.54 (m, 56H); ¹³C NMR (125.77 MHz, CDCl₃) δ 212.12,183.57, 174.57, 143.28, 122.32, 103.27, 101.72, 101.27, 86.07, 78.98,78.88, 76.68, 76.45, 76.04, 75.99, 75.28, 74.99, 65.56, 60.44, 54.06,49.52, 49.02, 46.47, 46.33, 41.56, 40.36, 39.76, 38.02, 36.29, 35.31,34.85, 32.78, 32.43, 31.66, 30.61, 26.67, 25.14, 24.42, 23.42, 20.41,17.05, 15.93, 12.15, 7.66, 7.55, 7.36, 7.25, 7.07, 6.98, 6.91, 6.04,5.81, 5.59, 5.50, 5.42, 5.40, 5.15, 4.58; FTIR (neat film) 2953, 2912,2877, 1722, 1460, 1414, 1378, 1239, 1163, 1103, 1007, 973, 825, 801, 738cm⁻¹; LRMS (ESI) m/z: Calcd for C₁₀₁H₁₉₈O₂₀Si₉Na (M+Na⁺) 2006.2, found2006.5.

S16 R_(f)=0.74 (benzene/ethyl acetate 4:1); characteristic resonancesfrom ¹H NMR (500 MHz, CDCl₃) δ 9.39 (s, 1H), 5.33 (s, 1H), 5.02 (d,J=2.2, 1H), 4.60 (s, 1H), 4.51 (s, 1H), 4.46 (m, 1H), 4.25 (d, J=7.3,1H), 4.15 (d, J=6.7, 1H), 3.92 (s, 1H), 3.83 (d, J=5.6, 1H), 3.77 (s,1H), 3.55 (m, 1H), 3.37 (d, J=8.7, 1H), 3.28 (m, 1H), 2.93 (dd, J=14.0,3.7, 1H), 2.20 (m, 1H), 1.36 (s, 3H), 1.17 (d, J=6.2, 3H), 1.14 (s, 3H),0.89 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 143.35, 122.24, 102.81, 99.43,97.18, 82.71, 77.86, 76.28, 75.83, 74.84, 74.05, 73.68, 73.09, 71.62,70.85, 70.49, 60.71, 54.47, 48.98, 48.43, 46.53, 41.54, 40.85, 39.83,38.06, 36.82, 36.10, 35.31, 34.81, 32.83, 32.32, 31.34, 30.66, 26.64,24.45, 23.60, 20.50, 18.42, 16.92, 16.12, 10.88, 7.42, 7.38, 7.30, 7.23,7.20, 7.17, 7.11, 7.04, 6.95, 5.62, 5.57, 5.47, 5.45, 5.40, 5.23, 5.15,5.11, 4.62; FTIR (neat film) 2953, 2913, 2876, 1724, 1459, 1414, 1379,1240, 1108, 1006, 974, 909, 885, 856, 821, 779, 738 cm⁻¹; LRMS (ESI)m/z: Calcd for C₁₀₂H₂₀₀O₂₀Si₉Na (M+Na⁺) 2020.3, found 2020.3.

O-(16-O-triethylsilyl-quillaic acid)4-O-triethylsilyl-[(2,3,4-tri-O-triethylsilyl-β-D-xylopyranosyl)-(1→3)]-[(2,3,4,6-tetra-O-triethylsilyl-β-D-galactopyranosyl-(1→2)]-β-D-glucuronosidebenzyl ester (21). To a solution of S14 (81.3 mg, 0.0409 mmol, 1.00equiv), tri-t-butylpyridine (102 mg, 0.412 mmol, 10.1 equiv) andpyridine (30 μL, 0.37 mmol, 9.1 equiv) in dichloromethane (0.68 mL) wasadded benzyl chloroformate (15 μL, 0.11 mmol, 2.6 equiv). After 6 h,additional benzyl chloroformate (3.0 μL, 0.021 mmol, 0.51 equiv) wasadded to the reaction. After stirring for 20 h, the reaction wasconcentrated and purified by silica gel chromatography (benzene/ethylacetate 1:0 to 24:1) to afford 21 (58.0 mg, 0.00279 mmol, 68% yield) asa white solid. R_(f)=0.69 (benzene/ethyl acetate 9:1); (500 MHz, CDCl₃)δ 9.70 (s, 1H, —CHO), 7.40-7.27 (m, 5H, aromatic), 5.34 (m, 1H,R₂C═CHR), 5.28 (d, J=12.5 Hz, 1H, PhCH ₂—), 5.09 (d, J=12.3 Hz, 1H, PhCH₂—), 4.55 (d, J=7.6 Hz, 1H), 4.53 (m, 1H), 4.42 (d, J=7.3 Hz, 1H), 4.12(d, J=7.6 Hz, 1H), 3.97-3.71 (m, 8H), 3.64-3.53 (m, 3H), 3.48 (m, 1H),3.39 (dd, J=9.3, 2.5 Hz, 1H), 3.37-3.31 (m, 2H), 3.25 (t, J=8.0 Hz, 1H),3.12 (t, J=10.9 Hz, 1H), 2.94 (dd, 1=14.0, 3.7 Hz, 1H), 2.21 (t, J=13.7Hz, 1H), 1.93-1.04 (m 20H), 1.35 (s, 3H, Me), 1.30 (s, 3H, Me),1.04-0.84 (m, 90H), 0.84-0.53 (m, 56H), see proton NMR below; ¹³C NMR(125.77 MHz, CDCl₃) δ 212.59, 182.49, 168.53, 143.44, 135.45, 128.62,128.43, 128.29, 122.31, 103.63, 101.55, 101.01, 86.22, 79.02, 78.90,76.61, 76.25, 76.02, 75.27, 75.12, 72.79, 72.67, 71.59, 71.22, 66.98,65.51, 60.47, 54.05, 49.58, 48.90, 46.52, 46.31, 41.61, 40.35, 39.76,38.07, 36.28, 35.31, 34.78, 32.81, 32.49, 31.68, 30.63, 26.68, 25.47,24.45, 23.43, 20.40, 16.97, 15.86, 12.29, 7.69, 7.59, 7.38, 7.27, 7.12,6.99, 6.92, 6.07, 5.81, 5.61, 5.53, 5.51, 5.43, 5.40, 5.16, 4.58; FTIR(neat film) 2954, 2914, 2877, 1754, 1723, 1705, 1459, 1414, 1379, 1240,1170, 1103, 1073, 1007, 972, 825, 801, 739 cm⁻¹; LRMS (ESI) m/z: Calcdfor C₁₀₈H₂₀₄O₂₀Si₉Na (M+Na⁺) 2096.3, found 2096.7.

Final Assembly of Semisynthetic Qs-7-Api

Fully-protected semisynthetic QS-7-Api (S15). A solution of borontrifluoride diethyl etherate (0.50 μL, 0.0040 mmol, 1.0 equiv) indichloromethane (20 μL) was added to a solution of the imidate 16 (8.3mg, 0.0041 mmol, 0.98 equiv) and 21 (12.7 mg, 0.00612 mmol, 1.50 equiv)in dichloromethane (0.50 mL) with 4 Å molecular sieves (86 mg) at −78°C. The reaction temperature was allowed to warm to 23° C. slowly, andtriethylamine (2.8 μL) was added after 14.5 h. The reaction wasconcentrated and purified by silica gel chromatography (silicapretreated with 0.1% triethylamine in benzene, then benzene/ethylacetate 99:1 to 24:1) to afford S15 (8.5 mg, 0.0033 mmol, 80% yield).R_(f)=0.52 (benzene/ethyl acetate 19:1); characteristic resonances from¹H NMR: (500 MHz, CDCl₃) δ 9.69 (s, 1H, —CHO), 7.49-7.04 (m, 50H,aromatic), 5.94 (s, 1H), 5.83 (s, 1H), 5.31 (d, J=7.2 Hz, 1H), 5.29-5.23(m, 2H), 5.12-5.06 (m, 2H), 5.01 (s, 1H), 4.93 (d, J=11.9 Hz, 1H),4.91-4.44 (m, 19H), 4.42 (d, J=7.3 Hz, 1H), 4.37 (d, J=4.7 Hz, 1H), 4.35(d, J=3.9 Hz, 1H), 4.23 (dd, J=2.5, 8.7 Hz, 1H), 4.19 (d, J=7.3 Hz, 1H),4.10-4.07 (m, 2H), 3.98-3.43 (m, 2H), 3.43-3.29 (m, 7H), 3.29-3.08 (m,6H), 2.97 (dd, J=13.8, 3.5 Hz, 1H), 2.83 (bs, 1H), 2.73 (bs, 1H), 2.10(s, 3H, —C(O)CH₃), 1.54 (s, 3H, Me), 1.40 (s, 3H, Me), 1.30 (s, 3H, Me),1.12 (s, 3H, Me); see proton NMR below; ¹³C NMR (125.77 MHz, CDCl₃) δ212.57, 176.57, 170.56, 168.54, 142.90, 139.36, 139.17, 138.90, 138.61,138.14, 138.02, 136.90, 135.46, 128.82, 128.61, 128.54, 128.43, 128.36,128.32, 128.27, 128.21, 128.07, 127.91, 127.80, 127.69, 127.62, 127.56,127.43, 127.39, 127.24, 127.04, 109.06, 107.31, 106.46, 103.64, 101.91,101.75, 101.56, 101.00, 99.70, 99.54, 93.81, 91.69, 87.28, 86.19, 84.99,82.50, 80.58, 79.16, 79.02, 78.90, 78.51, 76.61, 76.35, 76.24, 76.00,75.72, 75.44, 75.28, 74.82, 74.67, 74.41, 73.69, 73.34, 73.07, 72.96,72.86, 72.79, 72.66, 72.52, 71.57, 71.23, 70.26, 68.92, 68.48, 66.96,65.61, 63.99, 60.45, 54.05, 49.58, 48.92, 46.67, 46.26, 41.56, 40.14,38.19, 36.20, 34.50, 33.07, 32.68, 30.79, 29.85, 28.23, 26.68, 26.50,25.51, 24.74, 23.61, 22.85, 20.95, 20.44, 18.53, 17.83, 17.68, 16.67,16.06, 14.27, 12.36, 7.76, 7.60, 7.39, 7.30, 7.27, 7.11, 6.99, 6.92,6.07, 5.80, 5.60, 5.52, 5.43, 5.40, 5.17, 4.58; FTIR (neat film) 2953,2935, 2914, 2877, 1747, 1456, 1377, 1239, 1239, 1172, 1100, 1008, 824,735, 697 cm⁻¹.

Semisynthetic QS-7-Api (1). A solution of fully-protected semisyntheticQS-7-Api S15 (1.1 mg, 0.00028 mmol, 1.0 equiv) in tetrahydrofuran (1.0mL) and ethanol (1.0 mL) in a 10-mL round-bottom flask was charged with10% (dry basis) palladium on carbon, wet, Degussa type E101 NE/W (2.2mg, 0.00094 mmol, 3.7 equiv). The reaction suspension was stirred underhydrogen pressure (50 psi) for 15 h and then was filtered through a 0.45pm polyvinylidene fluoride filter disk. The filter was washed withmethanol (4 mL), and the filtrate and rinsing were concentrated in a25-mL round bottom flask to give a clear residue.

The reaction flask was cooled to 0° C. and charged with a pre-cooled (0°C.) solution of trifluoroacetic acid (1.0 mL, TFA/water 1:1). Afterstirring for 95 min, the reaction mixture was concentrated in vacuo at0° C. The resulting white solid residue was purified by RP-HPLC on anXBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a linear gradientof 32→37% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30 min at aflow rate of 5 mL/min. The fraction containing the major peak(t_(R)=14.85 min) was collected and lyophilized to dryness. A total ofthree deprotection runs were performed on S15 (3.3 mg, 0.00084 mmol) toafford semisynthetic QS-7-Api (1) (1.2 mg, 0.00064 mmol, 77% yield) as awhite solid. ¹H NMR (500 MHz, 7:3 D₂O:CD₃CN) δ 9.36 (s, 1H, —CHO), 5.39(d, J=8.0 Hz, 1H), 5.34 (m, 1H, R₂C═CHR), 5.22 (d, J=3.1 Hz, 1H), 5.12(d, J=2.9 Hz, 1H), 5.03 (s, 1H), 4.86 (s, 1H), 4.67 (d, J=7.7 Hz, 1H),4.63 (d, J=7.8 Hz, 1H), 4.55 (d, J=7.7 Hz, 1H), 4.50 (d, J=7.7 Hz, 1H),4.33 (m, 1H), 4.05 (d, J=10.1 Hz, 1H), 4.02 (m, 1H), 3.99-3.94 (m, 2H),3.92-3.57 (m, 22H), 3.55-3.45 (m, 7H), 4.33 (m, 1H), 3.42-3.15 (m, 12H),2.86 (dd, J=14.5, 3.6 Hz, 1H), 2.14 (s, 3H, —C(O)CH ₃), 1.89-1.30 (m,14H), 1.28 (s, 3H, Me), 1.22 (d, J=6.2 Hz, 3H, Me), 1.15 (d, J=6.1 Hz,3H, Me), 1.08 (s, 3H, Me), 1.05 (m, 1H), 1.02 (m, 1H), 0.99 (d, J=6.3Hz, 3H, Me), 0.93 (s, 3H, Me), 0.89 (s, 3H, Me), 0.84 (s, 3H, Me), 0.71(s, 3H, Me).

Example 2

This Example demonstrates that certain methods described above forExample 1 are applicable to substrates that differ in chemicalstructure.

Final Assembly of Semisynthetic Qs-21-Api

Fully protected QS-21-Api (140). A solution of boron trifluoride diethyletherate (0.49 μL, 0.0039 mmol, 0.50 equiv) in dichloromethane (10 μL)was added to a solution of imidate 23 (17.6 mg, 0.00780 mmol, 1.00equiv) and carboxylic acid 139 (24.3 mg, 0.0117 mmol, 1.50 equiv) indichloromethane (0.260 mL) with 4 Å molecular sieves (50 mg) at −78° C.The reaction temperature was allowed to warm to 23° C. slowly, andtriethylamine (20 μL) was added after 16 h. The reaction wasconcentrated and purified by silica gel chromatography (silicapretreated with 0.2% triethylamine in benzene, then benzene/ethylacetate 99:1 to 47:3) to afford 140 (13.8 mg, 0.0033 mmol, 42% yield) asa clear film. R_(f)=0.42 (benzene/ethyl acetate 19:1); characteristicresonances from ¹H NMR (500 MHz, CDCl₃) δ 9.68 (s, 1H), 5.96 (s, 1H),5.71 (s, 1H), 5.28 (d, J=12.4, 1H), 5.09 (d, J=12.4, 1H), 4.19 (d,J=7.2, 2H), 2.92 (dd, J=13.6, 3.7, 1H), 2.23 (t, J=13.5, 1H), 1.42 (s,3H), 1.34 (s, 3H), 1.29 (s, 3H), 1.26 (s, 3H), 1.22 (s, 3H); ¹³C NMR(126 MHz, CDCl₃) δ 171.03, 170.73, 168.51, 138.25, 138.12, 137.41,136.92, 135.43, 129.75, 128.65, 128.62, 128.57, 128.54, 128.43, 128.40,128.29, 128.26, 127.97, 127.93, 127.85, 127.81, 127.62, 127.32, 107.37,106.52, 91.72, 87.23, 85.97, 84.24, 81.34, 79.22, 79.03, 78.38, 76.62,76.20, 75.97, 75.28, 74.13, 73.72, 73.41, 73.10, 72.75, 71.92, 71.59,71.20, 67.00, 63.87, 63.27, 60.43, 54.11, 49.01, 43.58, 41.68, 40.86,40.04, 39.56, 36.98, 36.30, 32.96, 30.66, 29.88, 27.56, 26.55, 26.12,26.06, 25.94, 25.90, 25.09, 24.59, 24.23, 18.53, 18.10, 18.04, 18.02,17.87, 17.74, 17.28, 16.45, 14.66, 14.59, 12.35, 12.12, 7.70, 7.60,7.53, 7.39, 7.32, 7.30, 7.27, 7.12, 7.04, 6.99, 6.93, 6.07, 5.80, 5.61,5.53, 5.49, 5.42, 5.40, 5.18, 5.05, 4.58, −4.09, −4.29, −4.36, −4.38,−4.45, −4.57, −4.77, −5.07, −5.17; FTIR (neat film) 2954, 2935, 2877,1735, 1458, 1380, 1250, 1163, 1096, 1005, 836, 777, 737, 698 cm⁻¹; LRMS(ESI) m/z: Calcd for C₂₂₁H₃₈₂O₄₆Si₁₄Na₂ (M+2Na⁺⁺) 2105.2, found 2106.2.

QS-21-Api (4). Three solutions of fully protected second-generationQS-2-Api 140 (3×2.0 mg, 0.0014 mmol, 1.0 equiv) in tetrahydrofuran(3×1.0 mL) and ethanol (3×1.0 mL) in three 10-mL round-bottom flaskswere charged with 10% (dry basis) palladium on carbon, wet, Degussa typeE101 NE/W (3×3.8 mg, 0.0054 mmol, 3.7 equiv). The reaction suspensionswere stirred under hydrogen pressure (50 psi) for 23 h and then filteredthrough three 0.45 pm polyvinylidene fluoride filter disks. The filterswere washed with methanol, and the filtrate and rinsing wereconcentrated in three 25-mL round bottom flasks to give the partiallyprotected product as a clear residue.

The reaction flasks were cooled to 0° C. and charged with a pre-cooled(0° C.) solution of trifluoroacetic acid (3×1.0 mL, TFA/water 3:1).After stirring for 75 min, the reaction mixtures were concentrated invacuo at 0° C. The resulting white solid residue was purified by RP-HPLCon an XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a lineargradient of 35→45% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30min at a flow rate of 5 mL/min. The fractions containing the major peak(t_(R)=27.6 min) were collected and lyophilized to afford syntheticQS-21-Api (4) (1.4 mg, 0.00070 mmol, 49% yield) as a white solid. LRMS(ESI) m/z: Calcd for C₉₂H₁₄₇O₄₆ (M-H⁺) 1987.92, found 1988.33. The ¹HNMR spectrum of QS-21-Api (4) was found to be in agreement with thepreviously reported characterization.

Fully protected QS-21-Xyl (141). A solution of boron trifluoride diethyletherate (0.33 μL, 0.0026 mmol, 0.50 equiv) in dichloromethane (10 μL)was added to a solution of imidate 126 (12.4 mg, 0.00529 mmol, 1.00equiv) and carboxylic acid 139 (15.8 mg, 0.00761 mmol, 1.44 equiv) indichloromethane (0.176 mL) with 4 Å molecular sieves (62 mg) at −78° C.The reaction temperature was allowed to warm to 23° C. slowly, andtriethylamine (20 μL) was added after 21 h. The reaction wasconcentrated and purified by silica gel chromatography (silicapretreated with 0.5% triethylamine in benzene, then benzene/ethylacetate 99:1 to 97:3:3) to afford 141 in about 77% purity (14.2 mg,0.0026 mmol, 49% yield) as a clear film. R_(f)=0.49 (benzene/ethylacetate 19:1); characteristic resonances from ¹H NMR (500 MHz, CDCl₃) δ9.68 (s, 1H), 5.28 (d, J=12.4, 1H), 5.20 (d, J=1.6, 1H), 5.09 (d,J=12.3, 1H), 4.97 (m, 1H), 4.94 (d, J=11.5, 1H), 4.92 (d, J=7.8, 1H),4.47 (d, J=10.1, 1H), 4.42 (d, J=7.2, 1H), 4.37 (d, J=10.7, 1H), 4.18(d, J=7.1, 2H), 3.13 (t, J=10.8, 1H), 3.04 (t, J=10.9, 1H), 2.92 (dd,J=14.6, 3.9, 1H), 2.22 (t, J=13.8, 1H), 1.43 (s, 3H), 1.34 (s, 3H), 1.28(s, 3H), 1.26 (s, 3H), 1.22 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 175.39,171.03, 170.72, 168.51, 143.57, 138.90, 138.87, 138.67, 138.47, 138.38,137.42, 135.43, 128.62, 128.60, 128.51, 128.48, 128.44, 128.42, 128.34,128.29, 128.15, 128.12, 128.11, 127.97, 127.78, 127.73, 127.60, 109.74,107.38, 103.71, 103.28, 102.41, 101.56, 100.99, 93.65, 86.02, 84.20,84.00, 82.82, 82.79, 79.87, 79.20, 79.03, 78.92, 78.52, 78.35, 76.66,76.20, 76.00, 75.79, 75.73, 75.26, 75.15, 74.58, 74.45, 73.48, 73.26,72.81, 72.66, 72.01, 71.58, 71.25, 67.14, 66.96, 66.32, 65.54, 64.03,63.81, 63.28, 60.40, 54.10, 49.60, 48.99, 46.35, 43.65, 42.79, 41.69,40.91, 40.02, 39.52, 38.67, 37.00, 36.29, 32.90, 32.71, 30.65, 29.89,27.58, 26.61, 26.12, 26.06, 25.94, 25.90, 25.72, 25.03, 24.52, 24.23,18.55, 18.10, 18.07, 18.04, 18.02, 17.62, 17.25, 16.40, 16.05, 14.65,14.60, 12.35, 12.23, 12.08, 7.70, 7.60, 7.39, 7.32, 7.29, 7.27, 7.12,6.99, 6.93, 6.07, 5.81, 5.61, 5.53, 5.48, 5.42, 5.18, 5.03, 4.58, −4.08,−4.11, −4.30, −4.36, −4.38, −4.44, −4.57, −4.74, −5.07, −5.17; FTIR(neat film) 2955, 2877, 1735, 1458, 1380, 1250, 1165, 1091, 1006, 837,777, 735, 698 cm⁻¹; LRMS (ESI) m/z: Calcd for C₂₂₈H₃₉₀O₄₆Si₁₄Na₂(M+2Na⁺⁺) 2151.2, found 2151.6.

QS-21-Xyl (5). Three solutions of fully protected second-generationQS-2-Xyl 141 (3×2.0 mg, 0.0014 mmol, 1.0 equiv) in tetrahydrofuran(3×1.0 mL) and ethanol (3×1.0 mL) in three 10-mL round-bottom flaskswere charged with 10% (dry basis) palladium on carbon, wet, Degussa typeE101 NE/W (3×3.8 mg, 0.0054 mmol, 3.7 equiv). The reaction suspensionswere stirred under hydrogen pressure (50 psi) for 23 h and then filteredthrough three 0.45 pm polyvinylidene fluoride filter disks. The filterswere washed with methanol, and the filtrate and rinsing wereconcentrated in three 25-mL round bottom flasks to give the partiallyprotected product as a clear residue.

The reaction flasks were cooled to 0° C. and charged with a pre-cooled(0° C.) solution of trifluoroacetic acid (3×1.0 mL, TFA/water 3:1).After stirring for 75 min, the reaction mixtures were concentrated invacuo at 0° C. The resulting white solid residue was purified by RP-HPLCon an XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a lineargradient of 35→45% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30min at a flow rate of 5 mL/min. The fractions containing the major peak(t_(R)=27.7 min) were collected and lyophilized to afford syntheticQS-21-Xyl (5) (1.4 mg, 0.00070 mmol, 50% yield) as a white solid. LRMS(ESI) m % z: Calcd for C₉₂H₁₄₇O₄₆ (M-H⁺) 1987.92, found 1987.94. The ¹HNMR spectrum of QS-21-Xyl (5) was found to be in agreement with thepreviously reported characterization.

Example 3

3,6-Di-O-benzyl-4-azido-4-deoxy-D-galactal (218). Sodium hydroxide(0.115 g, 2.89 mmol, 0.357 equiv) was added to a solution of glycal 221(2.930 g, 8.063 mmol, 1.000 equiv) in methanol (40 mL) at 0° C., and thereaction was stirred at 23° C. After 14 h, the reaction was concentratedto a sticky tan solid, and trace solvent was removed by co-evaporationwith toluene (7 mL).

Dimethylformamide (40 mL) was added to the residue, and the resultingbrown suspension was cooled to 0° C. Sodium hydride (60% dispersion inoil, 0.977 g, 24.4 mmol, 3.03 equiv) was added to the reaction, followedby benzyl bromide (4.80 mL, 40.3 mmol, 5.01 equiv). After 3 h, theorange suspension was stirred at 23° C. for 16 h. The reaction wasquenched with methanol (20 mL), diluted with dichloromethane (100 mL),and washed with water (100 mL). The aqueous layer was extracted withdichloromethane (80 mL), and the combined organic layers were washedwith water (100 mL), dried with magnesium sulfate, and purified bysilica gel chromatography (hexane/ethyl acetate 9:1 to 4:1) to afford218 (2.199 g, 6.258 mmol, 78% yield) as a yellow oil. R_(f)=0.61(hexane/ethyl acetate 3:1); ¹H NMR (400 MHz, CDCl₃) δ 7.41-7.26 (m,10H), 6.37 (dd, J=6.3, 1.9, 1H), 4.83 (dt, J=6.4, 1.8, 1H), 4.70 (d,J=12.0, 1H), 4.63 (d, J=11.9, 1H), 4.61 (d, J=11.8, 1H), 4.55 (d,J=11.8, 1H), 4.41 (m, 1H), 4.08 (t, J=6.6, 1H), 3.97 (m, 1H), 3.70 (dd,J=6.6, 1.7, 2H; ¹³C NMR (126 MHz, CDCl₃) δ 144.69, 137.76, 137.69,128.68, 128.67, 128.14, 128.09, 128.04, 127.76, 101.00, 74.66, 73.84,71.57, 70.92, 68.84, 55.39; FTIR (neat film) 3031.5, 2868.5, 2109.9,1650.9, 1496.5, 1454.2, 1333.0, 1277.3, 1230.7, 1097.1, 1052.8, 1028.2,735.9, 697.6, 668.1 cm⁻¹; HRMS (ESI) m/z: Calcd for C₂₀NH₂₁N₃O₃Na(M+Na⁺) 374.1481, found 374.1479.

O-Acetyl2-O-acetyl-4-azido-4-deoxy-3,6-di-O-benzyl-β-D-galactopyranoside (222).Boron trifluoride diethyl etherate (74.0 μL, 0.636 mmol, 0.219 equiv)was added to a solution of glycal 218 (1.020 g, 2.904 mmol, 1.000 equiv)and iodobenzene diacetate (1.119 g, 3.474 mmol, 1.196 equiv) indichloromethane (36 mL) in a cold bath at −50° C. After 25 min, theyellow solution was transferred to a cold bath at −25° C. and stirredfor an additional 30 min. Triethyl amine (2.0 mL) was added, and theresulting suspension was diluted with dichloromethane (20 mL) and washedwith saturated aqueous sodium bicarbonate solution (30 mL). The aqueouslayer was extracted with dichloromethane (30 mL), and the combinedorganic layers were dried with magnesium sulfate and purified by silicagel chromatography (hexane/ethyl acetate 4:1 to 3:1) to afford 222(1.163 g, 2.477 mmol, 85% yield) as a yellow solid. R_(f)=0.25(hexane/ethyl acetate 3:1); ¹H NMR (400 MHz, CDCl₃) δ 7.41-7.28 (m,10H), 5.51 (d, J=8.3, 1H), 5.30 (dd, J=9.8, 8.4, 1H), 4.74 (d, J=12.2,1H), 4.56 (d, J=12.3, 1H), 4.54 (s, 2H), 4.08 (dd, J=3.6, 1.1, 1H), 3.74(td, J=6.7, 1.3, 1H), 3.69 (dd, J=9.8, 3.6, 1H), 3.63 (d, J=6.8, 2H),2.06 (s, 3H), 2.00 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 169.50, 169.38,137.51, 137.25, 128.73, 128.69, 128.32, 128.23, 128.20, 127.92, 92.47,78.73, 73.86, 72.60, 72.23, 69.78, 67.82, 59.10, 20.96, 20.88; FTIR(neat film) 2920.8, 2107.7, 1755.9, 1454.8, 1368.4, 1232.5, 1214.6,1059.1, 739.3, 698.4 cm⁻¹; HRMS (ESI) m/z: Calcd for C₂₄H₂₇N₃O₇Na(M+Na⁺) 492.1747, found 492.1755.

4-azido-4-deoxy-3,6-di-O-benzyl-D-galactopyranose (223). Potassiumcarbonate (0.600 g, 4.34 mmol, 3.99 equiv) was added to a solution ofdiacetate 222 (0.511 g, 1.09 mmol, 1.00 equiv) in methanol (50 mL) andwater (5 mL). After 1.5 h, the yellow solution was decanted from theundissolved potassium carbonate and concentrated to about 4 mL and thendiluted with dichloromethane (50 mL) and washed with saturated aqueoussodium bicarbonate solution (10 mL). The aqueous layer was extractedwith dichloromethane (2×10 mL) and the combined organic layers weredried with magnesium sulfate and purified by silica gel chromatography(ethyl acetate) to afford 223 (0.353 g, 0.917 mmol, 84% yield) as ayellow oil (1:1 α:β). R_(f)=0.17, 014 (hexane/ethyl acetate 1:1); ¹H NMR(400 MHz, CDCl₃) δ 7.43-7.27 (m, 20H), 5.28 (d, J=3.8, 1H), 4.78 (d,J=11.5, 1H), 4.78 (d, J=11.5, 1H), 4.67 (d, J=11.5, 1H), 4.68 (d,J=11.5, 1H), 4.58 (d, J=11.9, 1H), 4.56 (d, J=11.9, 1H), 4.52 (d,J=11.9, 1H), 4.51 (d, J=11.9, 1H), 4.51 (d, J=7.7, 1H), 4.21 (td, J=6.5,1.1, 1H), 4.02 (dd, J=3.4, 1.3, 1H), 3.99 (dd, J=9.9, 3.8, 2H), 3.85(dd, J=9.7, 3.5, 1H), 3.73 (dd, J=9.5, 7.7, 1H), 3.69-3.51 (m, 6H); ¹³CNMR (126 MHz, CDCl₃) δ 137.62, 137.50, 137.48, 128.67, 128.59, 128.16,128.13, 128.10, 128.09, 128.07, 128.02, 128.00, 97.08, 92.50, 80.85,78.06, 73.64, 73.61, 72.63, 72.51, 72.14, 71.82, 69.25, 68.76, 68.68,67.38, 60.27, 59.49; FTIR (neat film) 3401.3, 3032.4, 2921.6, 2105.6,1454.4, 1367.2, 1279.3, 1095.8, 1028.3, 738.1, 698.0 cm⁻¹; HRMS (ESI)m/z: Calcd for C₂₀H₂₃N₃O₅Na (M+Na⁺) 408.1535, found 408.1535.

O-triisopropylsilyl4-azido-4-deoxy-3,6-di-O-benzyl-β-D-galactopyranoside (224).Triisopropylsilyl chloride (0.63 mL, 3.0 mmol, 1.2 equiv) was added to asolution of hemiacetal 223 (0.959 g, 2.49 mmol, 1.00 equiv), imidazole(0.409 g, 6.01 mmol, 2.41 equiv), and 4-dimethylaminopyridine (29 mg,0.24 mmol, 0.096 equiv) in dimethylformamide (2.5 mL). After 19 h, theyellow solution was concentrated and purified by silica gelchromatography (hexane/ethyl acetate 19:1 to 9:1) to afford 224 (0.800g, 1.48 mmol, 59% yield) as a colorless oil. R_(f)=0.49 (hexane/ethylacetate 4:1); ¹H NMR (500 MHz, CDCl₃) δ 7.43-7.24 (m, 10H), 4.78 (d,J=11.8, 1H), 4.75 (d, J=11.8, 1H), 4.55 (d, J=11.6, 1H), 4.52 (d,J=12.0, 1H), 4.51 (d, J=7.3, 1H), 3.98 (d, J=3.6, 1H), 3.72 (ddd, J=9.5,7.3, 2.1, 1H), 3.69-3.58 (m, 3H), 3.56 (dd, J=9.6, 3.7, 1H), 2.26 (d,J=2.2, 1H), 1.19-0.99 (m, 21H); ¹³C NMR (126 MHz, CDCl₃) δ 137.86,137.84, 128.70, 128.63, 128.08, 128.05, 127.97, 127.93, 98.09, 80.69,73.81, 73.78, 72.80, 72.01, 68.83, 59.83, 17.96, 17.90, 12.33; FTIR(neat film) 3463.8, 2943.4, 2866.2, 2108.6, 1455.3, 1366.1, 1280.0,1185.4, 1099.0, 1028.7, 1014.6, 997.6, 883.5, 805.4, 736.3, 695.5, 669.0cm⁻¹; HRMS (ESI) m/z: Calcd for C₂₉H₄₃N₃O₅SiNa (M+Na⁺) 564.2870, found564.2870.

Azido tetrasaccharide 225. Trifluoromethanesulfonic anhydride (70 μL,0.42 mmol, 2.0 equiv) was added to a solution of trisaccharide 81 (0.168mg, 0.203 mmol, 1.00 equiv), phenyl sulfoxide (173 mg, 0.855 mmol, 4.20equiv) and 2,4,6-tri-tert-butylpyridine (262 mg, 1.06 mmol, 5.21 equiv)in dichloromethane (20 mL) at −78° C. The reaction stirred in a coldbath at −78° C. for 8 min and then was transferred to a bath between −55and −50° C. for 70 min. A solution of pyranoside 224 (137 mg, 0.253mmol, 1.24 equiv) in dichloromethane (3.0 mL) at −78° C. was added viacannula, and the reaction was warmed slowly from −50° C. to 0° C. over 4h and then stirred at 23° C. for 45 min. Triethylamine (1.0 mL) wasadded to the reaction mixture, which was concentrated and purified bysilica gel chromatography (benzene/ethyl acetate 49:1 to 9:1) to affordtetrasaccharide 225 (185 mg, 0.137 mmol, 67% yield) as a clear film.R_(f)=0.59 (benzene/ethyl acetate 9:1); ¹H NMR (500 MHz, CDCl₃) δ7.53-7.18 (m, 30H), 5.95 (s, 1H), 5.67 (s, 2H), 4.89 (d, J=7.6, 1H),4.86 (d, J=11.1, 1H), 4.72 (d, J=11.6, 1H), 4.63-4.47 (m, 8H), 4.44 (d,J=11.8, 1H), 4.41 (s, 1H), 4.13 (dd, J=7.5, 5.7, 1H); 4.04 (d, J=5.6,1H), 4.01 (dd, J=3.4, 0.8, 1H), 3.97-3.83 (m, 3H), 3.93 (s, 2H), 3.79(t, J=9.0, 1H), 3.69-3.53 (m, 7H), 3.36 (td, J=9.6, 5.3, 1H), 3.24 (dd,J=9.1, 7.6, 1H), 3.14 (dd, J=11.6, 10.0, 1H), 1.48 (s, 3H), 1.34 (s,3H), 1.23 (d, J=6.2, 3H), 1.10-0.95 (m, 21H); ¹³C NMR (126 MHz, CDCl₃) δ138.22, 138.12, 137.76, 137.01, 136.95, 129.72, 128.89, 128.79, 128.74,128.63, 128.56, 128.52, 128.41, 128.38, 128.32, 128.32, 128.09, 128.01,127.92, 127.83, 127.75, 127.59, 127.32, 109.15, 107.30, 106.46, 102.56,97.27, 97.13, 91.65, 87.15, 82.33, 81.20, 78.43, 78.28, 78.49, 76.74,76.26, 74.06, 73.99, 73.81, 73.66, 73.33, 72.97, 71.74, 71.67, 71.15,68.68, 64.51, 63.78, 58.89, 27.95, 26.60, 18.07, 17.99, 17.93, 12.43;FTIR (neat film) 2927.8, 2866.8, 2108.8, 1455.1, 1367.0, 1184.5, 1092.1,990.3, 735.7, 697.8 cm⁻¹; HRMS (ESI) m/z: Calcd for C₇₆H₉₅N₃O₁₇SiNa(M+Na⁺) 1372.6328, found 1382.6343.

Azido tetrasaccharide hemiacetal 226. Tetrabutylammonium fluoridesolution (1.0 M in tetrahydrofuran, 14 μL, 0.014 mmol, 1.0 equiv) wasadded to a solution of triisopropylsilyl acetal 225 (18.5 mg, 0.0137mmol, 1.00 equiv) in tetrahydrofuran (1.8 mL) at 0° C. After 17 min,methanol (1.0 mL) was added, and the solvent was removed in vacuo at 0°C. to give a pale yellow oil that was purified by silica gelchromatography (benzene/ethyl acetate 19:1 to 9:1) to afford 226 (15.2mg, 0.0127 mmol, 93% yield). R_(f)=0.61 (benzene/ethyl acetate 4:1);characteristic resonances from ¹H NMR (500 MHz, CDCl₃) δ 7.54-7.20 (m,30H), 5.96 (s, 1H), 5.69 (s, 1H), 4.87 (dd, J=12.2, 7.5, 1H), 4.83 (d,J=11.4, 1H), 4.72 (d, J=11.7, 1H), 3.38 (m, 1H), 3.26 (m, 1H), 3.16 (m,1H); FTIR (neat film) 3426.0, 2933.1, 2107.1, 1454.6, 1370.7, 1220.2,1092.6, 1027.0, 992.4, 735.5, 698.3 cm⁻¹, HRMS (ESI) m/z: Calcd forC₆₇H₇₅N₃O₁₇Na (M+Na⁺) 1216.4994, found 1216.4939.

Azido tetrasaccharide trichloroacetimidate 227. Trichloroacetonitrile(191 μL, 1.91 mmol, 150 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene(7.6 μL, 0.051 mmol, 4.0 equiv) were added to a solution of hemiacetal226 (15.2 mg, 0.0127 mmol, 1.00 equiv) at 0° C. After 14 h at 0° C. and45 min at 23° C., the solution was concentrated and purified by silicagel chromatography (benzene/ethyl acetate 19:1 to 4:1) to afford 227(16.2 mg, 0.0121 mmol, 95% yield) as a clear film. R_(f)=0.50(benzene/ethyl acetate 9:1); characteristic peaks from ¹H NMR (500 MHz,CDCl₃) δ 8.52 (s, 1H), 7.59-7.14 (m, 30H), 5.95 (s, 1H), 5.67 (s, 1H),5.23 (s, 1H), 4.88 (d, J=7.5, 1H), 4.79 (d, J=10.8, 1H), 4.77 (d,J=11.2, 1H), 4.67 (d, J=11.7, 1H), 4.20 (dd, J=10.0, 3.5, 1H), 3.83 (dd,J=11.5, 5.5, 2H), 3.78 (t, J=9.0, 1H), 3.33 (m, 1H), 3.21 (dd, J=8.9,7.8, 1H), 3.13 (dd, J=11.7, 9.9, 1H), 1.21 (d, J=6.0, 3H); ¹³C NMR (126MHz, CDCl₃) δ 161.14, 138.18, 138.09, 138.01, 137.63, 137.48, 136.89,129.76, 128.70, 128.64, 128.56, 128.52, 128.46, 128.39, 128.21, 128.14,128.11, 128.09, 127.95, 127.92, 127.84, 127.60, 127.31, 110.02, 109.41,107.24, 106.55, 106.48, 101.83, 99.71, 95.88, 95.45, 91.73, 91.67,91.19, 87.17, 80.69, 78.23, 77.26, 76.67, 76.60, 76.02, 74.68, 73.83,73.81, 73.69, 73.36, 73.04, 72.68, 71.13, 69.97, 68.26, 64.96, 63.77,60.35, 27.88, 26.55, 17.84, 17.60, 12.42; FTIR (neat film) 3031.0,2932.9, 2110.8, 1671.8, 1454.8, 1368.6, 1280.7, 1242.2, 1220.8, 1095.6,1020.2, 991.5, 912.4, 860.5, 795.0, 735.0, 698.1, 677.1 cm⁻¹; HRMS (ESI)m/z: Calcd for C₆₉H₇₅Cl₃N₄O₁₇Na (M+Na⁺) 1359.4091, found 1359.4127.

Azido saponin 228. Boron trifluoride diethyl etherate (2.0 μL, 0.016mmol, 0.51 equiv) was added to a solution of imidate 227 (41.7 mg,0.0311 mmol, 1.00 equiv) and carboxylic acid 139 (88.6 mg, 0.0427 mmol,1.37 equiv) with 4 Å molecular sieves (147 mg) in dichloromethane (1.04mL) at −78° C. After 3 min, the reaction flask was transferred to a −43°C. cold bath (11:10 ethanol:water/CO₂), and the reaction temperature wasallowed to warm slowly to 23° C. Triethylamine (0.20 mL) was added after14.5 h, and the reaction was concentrated and purified by silica gelchromatography (silica pretreated with 0.2% triethylamine in benzene,then benzene/ethyl acetate 49:1 to 9:1) to afford 228 (83.5 mg, 0.0257mmol, 82% yield) as a white solid. R_(f)=0.58 (benzene/ethyl acetate9:1); characteristic resonances from ¹H NMR (500 MHz, CDCl₃) δ 9.68 (s,1H), 5.95 (s, 111), 5.70 (s, 111), 5.17 (d, J=2.4, 111), 5.08 (d,J=12.4, 1H), 4.83 (d, J=11.2, 1H), 4.79 (d, J=7.6, 1H), 4.71 (d, J=11.1,1H), 4.61 (d, J=11.1, 1H), 4.18 (d, J=7.3, 1H), 4.11 (dd, J=6.4, 2.7,1H), 3.24 (m, 2H), 3.13 (m, 2H), 2.88 (dd, J=14.1, 3.7, 1H), 2.20 (t,J=13.5, 1H), 1.40 (s, 3H), 1.32 (s, 3H), 1.29 (s, 3H), 1.22 (s, 3H),1.15 (d, J=6.2, 3H), 0.85 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 212.45,175.31, 168.49, 143.39, 138.22, 138.13, 138.09, 137.64, 137.11, 136.89,135.42, 129.73, 128.70, 128.63, 128.60, 128.57, 128.55, 128.53, 128.43,128.40, 128.38, 128.27, 128.12, 128.04, 127.98, 127.96, 127.91, 127.83,127.78, 127.60, 127.30, 121.97, 109.70, 107.32, 106.50, 103.64, 102.30,101.56, 100.99, 97.96, 93.89, 91.70, 87.20, 86.20, 81.33, 80.93, 78.97,78.88, 78.77, 78.28, 76.77, 76.58, 75.99, 75.25, 74.12, 73.73, 73.70,73.37, 73.08, 72.77, 72.68, 72.02, 71.57, 71.20, 71.15, 67.67, 67.60,66.96, 65.49, 63.81, 60.42, 59.03, 54.06, 49.52, 49.04, 46.75, 46.29,41.60, 40.83, 39.91, 38.12, 36.23, 35.32, 34.89, 32.87, 32.60, 30.88,30.57, 29.87, 27.53, 26.50, 25.78, 25.48, 24.44, 23.44, 20.37, 17.73,17.29, 15.97, 12.30, 7.68, 7.58, 7.47, 7.37, 7.31, 7.27, 7.25, 7.10,6.97, 6.91, 6.05, 5.79, 5.59, 5.51, 5.48, 5.41, 5.38, 5.14, 5.03, 4.57;FTIR (neat film) 2952.9, 2912.2, 2876.4, 2106.7, 1752.4, 1497.1, 1456.5,1379.5, 1240.2, 1165.7, 1098.7, 1006.1, 913.3, 863.1, 824.5, 736.4,697.5 cm⁻¹; LRMS (ESI) m % z: Calcd for C₁₇₆H₂₇₉N₃O₃₅Si₉Na₂ (M+2Na⁺)1646.39, found 1648.64.

Amino saponin 229. A yellow solution of diphenyldiselenide (45.8 mg,0.147 mmol, 1.00 equiv) and hypophosphorous acid, 50% in water (0.16 mL,1.6 mmol, 11 equiv) in tetrahydrofuran (2.0 mL) was heated at 40° C. for40 min until it turned colorless. The solution was then removed from theheat, diluted with benzene (2.0 mL) and deionized water (2.0 mL). Thelower phase of the resulting biphasic suspension was removed by syringe(2.4 mL), and the remaining organic layer was dried with sodiumsulphate.

This freshly prepared solution of phenylselenol (1.7 mL, ˜0.14 mmol, 30equiv) was added to a solution of azido saponin 228 (15.0 mg, 0.00461mmol, 1.00 equiv) in triethylamine (5.0 mL) and was heated at 30° C. for15 h.

A second solution of phenylselenol was prepared by heatingdiphenyldiselenide (53.7 mg, 0.172 mmol, 1.00 equiv) and hypophosphorousacid, 50% in water (0.19 mL, 1.8 mmol, 11 equiv) in tetrahydrofuran (2.0mL) was heated at 40° C. for 30 min until it turned colorless. Thissolution of phenylselenol was then removed from the heat, diluted withbenzene (2.0 mL) and deionized water (2.0 mL). The lower phase of theresulting biphasic suspension was removed by syringe (2.4 mL), and theremaining organic layer was dried with sodium sulphate.

The second solution of phenylselenol (1.5 mL, ˜0.14 mmol, 30 equiv) wasadded to the reaction flask containing azido saponin 228, which washeated at 40° C. for 8 h. Additional phenylselenol solution (0.6 mL,˜0.06 mmol, 12 equiv) was added, and the reaction was stirred at 40° C.for an additional 2 h. The reaction mixture was concentrated andpurified by silica gel chromatography (benzene to 3:2 benzene/ethylacetate) to afford 228 (13.6 mg, 0.00422 mmol, 91% yield) as a whitesolid film. R_(f)=0.24 (benzene/ethyl acetate 9:1); characteristicresonances from ¹H NMR (500 MHz, CDCl₃) δ 9.69 (s, 1H), 5.96 (s, 1H),5.71 (s, 1H), 5.37 (d, J=7.8, 1H), 5.31 (m, 1H), 5.28 (d, J=12.4, 1H),5.19 (d, J=1.7, 1H), 5.09 (d, J=12.4, 1H), 4.83 (d, J=11.1, 1H), 4.82(d, J=7.5, 1H), 4.64 (t, J=10.9, 2H), 4.59 (d, J=1.8, 2H), 4.18 (d,J=7.3, 1H), 3.25 (t, J=8.4, 2H), 3.14 (m, 2H), 2.89 (dd, J=14.5, 4.0,1H), 1.42 (s, 3H), 1.33 (s, 3H), 1.29 (s, 3H), 1.24 (s, 3H), 1.16 (d,J=6.2, 3H); FTIR (neat film) 2952.7, 2912.3, 2876.2, 1753.9, 1453.9,1097.9, 1006.5, 862.44, 824.6, 737.1, 697.1, 668.4 cm⁻¹.

Fully protected compound I-9 (248). A solution of ethyl chloroformate(0.45 μL, 0.0047 mmol, 1.2 equiv) in tetrahydrofuran (10 μL) was addedto a solution of carboxylic acid 243 (5.0 mg, 0.0047 mmol, 1.2 equiv)and triethylamine (0.82 μL, 0.0059 mmol, 1.5 equiv) in tetrahydrofuran(1.0 mL) at 0° C. After 2.5 h, amino saponin 229 (12.7 mg, 0.00394 mmol,1.00 equiv) was added in a solution of tetrahydrofuran (3.0 mL). Thereaction was warmed slowly to 10° C. over 12 h and stirred at 10° C. foran additional 2 h. The reaction was concentrated and purified by silicagel chromatography (hexane/ethyl acetate 4:1) to afford 248 (15.6 mg,0.00365 mmol, 93% yield) as a clear film. R_(f)=0.30 (hexane/ethylacetate 4:1); characteristic resonances from ¹H NMR (500 MHz, CDCl₃) δ9.69 (s, 1H), 6.31 (d, J=9.0, 1H), 6.25 (d, J=9.9, 1H), 5.96 (s, 1H),5.72 (s, 1H), 5.37 (d, J=7.3, 1H), 5.34 (m, 1H), 5.28 (d, J=12.4, 1H),5.18 (s, 1H), 5.09 (m, 1H), 4.85 (d, J=11.1, 1H), 4.82 (d, J=10.6, 1H),4.78 (d, J=7.6, 1H), 4.37 (d, J=10.4, 1H), 4.23 (m, 1H), 4.18 (d, J=7.2,1H), 3.25 (m, 2H), 3.13 (td, J=11.2, 3.8, 2H), 2.90 (dd, J=14.3, 2.8,1H), 2.22 (t, J=13.3, 1H), 1.44 (s, 3H), 1.34 (s, 3H), 1.29 (s, 3H),1.20 (s, 3H), 1.11 (d, J=6.1, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.74,170.67, 168.50, 138.25, 138.12, 138.10, 138.01, 137.76, 136.91, 135.44,129.75, 128.65, 128.62, 128.57, 128.54, 128.48, 128.46, 128.43, 128.40,128.29, 127.97, 127.92, 127.84, 127.66, 127.62, 127.31, 109.73, 107.38,107.02, 106.52, 91.72, 87.22, 86.49, 86.21, 84.14, 81.41, 79.17, 76.02,74.21, 73.72, 73.54, 73.39, 73.12, 71.22, 71.17, 67.74, 66.98, 63.90,63.85, 63.41, 54.07, 50.12, 48.85, 41.61, 39.88, 38.68, 36.80, 36.26,32.85, 30.57, 29.85, 27.52, 26.57, 25.67, 25.38, 24.40, 18.56, 18.21,18.13, 18.06, 18.02, 17.69, 17.14, 15.98, 15.04, 14.35, 12.30, 12.07,7.70, 7.60, 7.39, 7.33, 7.31, 7.27, 7.11, 6.99, 6.93, 6.07, 5.81, 5.60,5.53, 5.49, 5.42, 5.40, 5.04, 4.59, −3.85, −3.99, −4.37, −4.42, −4.45,−4.57, −4.68, −5.10, −5.17; FTIR (neat film) 2954.1, 2933.4, 2877.0,2858.3, 1752.3, 1675.5, 1458.6, 1379.5, 1251.0, 1099.8, 836.6, 777.8,737.0, 697.0, 668.5 cm⁻¹; LRMS (ESI) m/z: Calcd for C₂₂₈H₃₉₀N₂O₄₅Si₁₄Na₂(M+2Na⁺⁺) 2157.24, found 2157.27.

Compound I-9. Three solutions of fully protected I-9 248 (3×2.0 mg,0.0014 mmol, 1.0 equiv) in tetrahydrofuran (3×1.0 mL) and ethanol (3×1.0mL) in three 10-mL round bottom flasks were charged with 10% (dry basis)palladium on carbon, wet, Degussa type E101 NE/W (3×3.8 mg, 0.0054 mmol,3.8 equiv). The three parallel reactions were stirred under hydrogenpressure (50 psi) for 24 h, and then the suspensions were each filteredthrough a 0.45 pm polyvinylidene fluoride filter disk, washed withmethanol (5 mL), and concentrated in a 25-mL round bottom flask.

A pre-cooled (0° C.) solution of trifluoroacetic acid (1.0 mL, TFA/water3:1) was added to each flask. After vigorous stirring for 75 min, thethree parallel reactions were concentrated in vacuo for 1 h at 0° C. togive white solid residue. This crude product was purified by RP-HPLC onan XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a lineargradient of 30→50% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30min at a flow rate of 5 mL/min. The fraction containing the major peak(t_(R)=12.15 min) was collected and lyophilized to dryness to affordcompound I-9 (2.4 mg, 85% yield) as a white solid. Characteristicresonances from ¹H NMR (500 MHz, 7:3 D₂O:CD₃CN) δ 9.36 (s, 1H), 5.34 (m,1H), 5.31 (d, J=7.9, 1H), 5.19 (d, J=3.1, 1H), 5.17 (d, J=1.5, 1H), 4.98(d, J=2.1, 1H), 4.67 (d, J=7.8, 1H), 4.56 (d, J=7.8, 1H), 4.51 (d,J=7.7, 1H), 4.28 (d, J=4.6, 1H), 4.06 (d, J=10.1, 1H), 2.87 (dd, J=14.6,3.3, 1H), 2.43 (d, J=6.6, 1H), 2.39 (dd, J=7.5, 13.8, 1H), 2.33 (dd,J=14.0, 5.3, 1H), 1.29 (s, 3H), 1.24 (d, J=6.0, 3H), 1.08 (s, 3H), 0.94(s, 3H), 0.90 (s, 3H), 0.68 (s, 3H); LRMS (ESI) m/z: Calcd forC₉₂H₁₄₉N₂O₄₅ (M-H⁺) 2001.94, found 2002.12.

Fully protected compound I-10 (249). A solution of ethyl chloroformate(0.30 μL, 0.0031 mmol, 2.0 equiv) in tetrahydrofuran (10 μL) was addedto a solution of carboxylic acid 247 (2.1 mg, 0.0030 mmol, 2.0 equiv)and triethylamine (0.45 μL, 0.0033 mmol, 2.1 equiv) in tetrahydrofuran(1.0 mL) at 0° C. After 1.5 h, amino saponin 229 (5.0 mg, 0.0016 mmol,1.0 equiv) was added in a solution of tetrahydrofuran (3.0 mL). Thereaction was warmed slowly to 12° C. over 14 h, concentrated, andpurified by silica gel chromatography (benzene to benzene/ethyl acetate47:3) to afford 249 (5.4 mg, 89% yield) as a clear film. R_(f)=0.64(benzene/ethyl acetate 9:1); characteristic resonances from ¹H NMR (500MHz, CDCl₃) δ 9.69 (s, 1H), 5.96 (s, 1H), 5.71 (s, 1H), 5.62 (m, 1H),5.39 (d, J=6.6, 1H), 5.28 (d, J=12.4, 1H), 5.19 (s, 1H), 5.09 (d,J=12.4, 1H), 4.85 (d, J=11.2, 1H), 4.82 (d, J=7.6, 1H), 4.79 (d, J=10.6,1H), 4.75 (d, J=1.6, 1H), 4.64 (d, J=11.2, 1H), 4.59 (d, J=1.7, 2H),4.18 (d, J=7.3, 1H), 3.25 (m, 2H), 3.14 (dd, J=19.6, 10.2, 2H), 2.88(dd, J=14.1, 3.5, 1H), 2.21 (t, J=13.4, 1H), 2.14 (t, J=7.4, 1H), 1.43(s, 3H), 1.34 (s, 3H), 1.30 (s, 3H), 1.23 (s, 3H), 1.12 (d, J=6.1, 3H);¹³C NMR (126 MHz, CDCl₃) δ 138.24, 138.11, 137.64, 136.91, 135.44,129.77, 128.64, 128.62, 128.57, 128.55, 128.51, 128.49, 128.45, 128.41,128.29, 127.98, 127.93, 127.85, 127.62, 127.32, 109.64, 108.54, 107.38,106.53, 91.72, 87.23, 84.34, 84.25, 81.46, 79.00, 78.89, 78.74, 78.67,78.24, 74.25, 73.73, 73.65, 73.40, 73.11, 71.84, 71.57, 71.20, 67.87,66.99, 63.87, 63.11, 54.03, 46.21, 41.71, 39.94, 37.06, 36.23, 32.86,32.08, 30.60, 29.93, 29.88, 29.86, 29.81, 29.72, 29.67, 29.59, 29.46,27.62, 26.53, 26.39, 26.08, 25.96, 25.90, 24.47, 22.86, 20.40, 18.53,18.04, 18.00, 17.72, 17.32, 16.04, 14.27, 12.33, 7.70, 7.60, 7.39, 7.32,7.29, 7.27, 7.12, 6.99, 6.93, 6.08, 5.81, 5.61, 5.53, 5.50, 5.42, 5.40,5.08, 4.58, 1.19, −4.18, −4.38, −4.54, −4.64, −5.03, −5.16; FTIR (neatfilm) 2952.9, 2929.0, 2876.6, 2856.9, 1750.9, 1734.2, 1457.5, 1241.6,1219.9, 1099.2, 1006.0, 836.7, 737.2, 697.5 cm⁻¹; LRMS (ESI) m/z: Calcdfor C₂₁₀H₃₅₁NO₄₂Si₁₁Na₂ (M+2Na⁺⁺) 1970.62, found 1971.83.

Compound I-10. Fully protected I-10 (249) (5.4 mg, 0.0014 mmol, 1.0equiv) was equally divided into three 10-mL round bottom flasks anddissolved in tetrahydrofuran (3×1.0 mL) and ethanol (3×1.0 mL). Thethree parallel reactions were charged with 10% (dry basis) palladium oncarbon, wet, Degussa type E101 NE/W (3×3.2 mg, 0.0045 mmol, 3.3 equiv)and stirred under hydrogen pressure (50 psi) for 24.5 h. The threesuspensions were combined and filtered through two 0.45 μmpolyvinylidene fluoride filter disks, washed with methanol (5 mL), andconcentrated in two 25-mL round bottom flasks.

A pre-cooled (0° C.) solution of trifluoroacetic acid (1.0 mL, TFA/water3:1) was added to both flasks. After vigorous stirring for 60 min, thetwo parallel reactions were concentrated in vacuo for 2 h at 0° C. toafford white solid residue. This crude product was purified by RP-HPLCon an XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a lineargradient of 30→40% acetonitrile (0.05% TFA) in water (0.05% TFA) over 17min at a flow rate of 5 mL/min. The fraction containing the major peak(t_(R)=15.5 min) was collected and lyophilized to dryness to affordcompound I-10 (2.0 mg, 78% yield) as an amorphous white solid.

Characteristic resonances from ¹H NMR (500 MHz, 7:3 D₂O:CD₃CN) δ 9.36(s, 4H), 5.34 (m, 1H), 5.32 (d, J=7.9, 1H), 5.19 (d, J=3.1, 1H), 5.17(d, J=1.4, 1H), 4.87 (d, J=1.8, 1H), 4.67 (d, J=7.7, 1H), 4.56 (d,J=7.8, 1H), 4.52 (d, J=7.9, 1H), 4.24 (d, J=4.2, 1H), 4.06 (d, J=10.1,1H), 3.96 (d, J=3.1, 1H), 2.89 (dd, J=14.3, 3.5, 1H), 1.08 (s, 3H), 0.94(s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.68 (s, 3H); LRMS (ESI) m/z: Calcdfor C₉₆H₁₃₈NO₄₂ (M-H⁺) 1856.87, found 1857.03.

Fully protected compound I-8 (251). Lauroyl chloride (6.6 μL, 0.029mmol, 12 equiv) was added to a solution of amine 229 (7.7 mg, 0.0024mmol, 1.0 equiv) and tri-tert-butylpyridine (52.5 mg, 0.212 mmol, 89equiv) in dichloromethane (10 mL). After 16 h, the reaction wasconcentrated and purified by silica gel chromatography (benzene tobenzene/ethyl acetate 4:1) to afford 251 (8.0 mg, 0.0023 mmol, 98%yield) as a clear film. R_(f)=0.58 (benzene/ethyl acetate 9:1);characteristic resonances from ¹H NMR (500 MHz, CDCl₃) δ 9.70 (s, 1H),5.96 (s, 1H), 5.71 (s, 1H), 5.62 (m, 1H), 5.38 (d, J=6.9, 1H), 5.30 (m,1H), 5.28 (d, J=12.4, 1H), 5.20 (d, J=1.6, 1H), 5.09 (d, J=12.4, 1H),4.64 (d, J=11.2, 1H), 4.59 (d, J=1.8, 2H), 4.18 (d, J=7.3, 1H), 3.25 (m,2H), 3.14 (m, 2H), 2.88 (dd, J=13.5, 3.0, 1H), 2.21 (t, 0.1=13.8, 1H),2.15 (t, J=7.1, 1H), 1.44 (s, 3H), 1.12 (d, J=6.1, 3H); ¹³C NMR (126MHz, CDCl₃) δ 138.25, 128.64, 128.61, 128.57, 128.55, 128.52, 128.48,128.45, 128.41, 128.29, 127.98, 127.92, 127.85, 127.83, 127.62, 127.32,112.24, 91.72, 74.25, 73.73, 73.64, 73.10, 63.86, 54.03, 46.16, 39.96,36.22, 32.85, 32.08, 32.05, 31.02, 30.60, 30.43, 29.84, 29.76, 29.74,29.61, 29.59, 29.53, 29.47, 29.39, 29.22, 27.61, 25.93, 25.51, 24.46,22.84, 19.68, 17.73, 14.28, 7.69, 7.60, 7.39, 7.31, 7.27, 7.11, 6.99,6.92, 6.07, 5.80, 5.60, 5.53, 5.49, 5.42, 5.39, 5.08, 4.58; FTIR (neatfilm) 2952.9, 2934.8, 2876.3, 1749.9, 1698.43, 1457.2, 1375.7, 1239.9,1098.5, 1006.4, 697.5, 669.4 cm⁻¹; LRMS (ESI) m/z: Calcd forC₁₈₇H₃₀₁NO₃₇Si₉Na₂ (M+2Na⁺⁺) 1725.47, found 1723.82.

Compound I-8. Three parallel solutions of fully protected I-8 (251)(3×1.25 mg, 0.0011 mmol, 1.0 equiv) in tetrahydrofuran (3×1.0 mL) andethanol (3×1.0 mL) in 10-mL round bottom flasks were charged with 10%(dry basis) palladium on carbon, wet, Degussa type E101 NE/W (3×2.3 mg,0.0054 mmol, 2.9 equiv). The three parallel reactions were stirred underhydrogen pressure (50 psi) for 26 h, and then the suspensions werecombined and filtered through two 0.45 pm polyvinylidene fluoride filterdisks, washed with methanol (5 mL), and concentrated in two 25-mL roundbottom flasks.

A pre-cooled (0° C.) solution of trifluoroacetic acid (1.0 mL, TFA/water1:1) was added to both parallel reactions. After vigorous stirring for35 min, the reactions were concentrated in vacuo at 0° C. to give whitesolid residue. This crude product was purified by RP-HPLC on an XBridgePrep BEH300 C18 column (5 μm, 10×250 mm) using a linear gradient of50→65% acetonitrile (0.05% TFA) in water (0.05% TFA) over 30 min at aflow rate of 5 mL/min. The product eluted as a broad peak (t_(R)=12.78min), and this fraction was collected and lyophilized to dryness toafford compound I-8 (1.3 mg, 0.00076 mmol, 69% yield) as an amorphoussolid. Characteristic resonances from ¹H NMR (500 MHz, 1:1 D₂O:CD₃CN) δ9.36 (s, 3H), 5.31 (m, 1H), 5.28 (d, J=7.7, 1H), 5.19 (s, 1H), 5.17 (d,J=3.1, 1H), 4.66 (d, J=7.8, 1H), 4.53 (d, J=7.8, 1H), 4.48 (d, J=7.9,1H), 4.40 (d, J=7.7, 1H), 4.05 (d, J=10.1, 2H), 3.95 (d, J=3.1, 1H),2.87 (m, 2H), 1.08 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.67 (s, 3H);LRMS (ESI) m/z: Calcd for C₈₁H₁₃₀NO₃₇ (M-H⁺) 1708.83, found 1709.37.

Example 4

Preclinical Evaluation of Synthetic QS-21 with GD3-KLH Conjugate Vaccine

This example demonstrates the in vivo immunogenicity of certaincompounds of the present invention. Using similar protocols, theimmuno-potentiating properties of the synthetic adjuvants SQS-21-Api andSQS-21-Xyl were evaluated in mice (C57BL/6J, female, six weeks of age).Although our previous synthetic chemistry efforts had unambiguouslyverified the chemical structure of SQS-21-Api (Kim, Y.-J.; Wang, P.;Navarro-Villalobos, M., Rohde, B. D.; Derryberry, J.; Gin, D. Y. J. Am.Chem. Soc. 2006, 128, 11906-11915) and SQS-21-Xyl (Deng, K.; Adams, M.M.; Damani, P.; Livingston, P. O.; Ragupathi, G.; Gin, D. Y. Angew.Chem., Int. Ed. 2008, 47, 6395-6398) as being identical to that of theprincipal constituents within NQS-21 (Wang, P.; Kim, Y.-J.;Navarro-Villalobos, M.; Rohde, B. D.; Gin, D. Y. J. Am. Chem. Soc. 2005,127, 3256-3257; Jacobsen, N. E.; Fairbrother, W. J.; Kensil, C. R.; Lim,A.; Wheeler, D. A.; Powell, M. F. Carbohydr. Res. 1996, 280, 1-14), thisexperiment was undertaken to evaluate the synthetic saponins to verifytheir biological activity given the variable heterogeneity incomposition within naturally derived NQS-21 (Note: NQS-21=naturallyderived QS-21; SQS-21-Mix=synthetic QS-21; SQS-21-Api=syntheticQS-21-Api; SQS-21-Xyl=synthetic QS-21-Xyl; SQS-7=synthetic QS-7). NQS-21is isolated as a 65:35 mixture of its Apiose and Xylose isomeric forms,yet it is extremely difficult to separate these constituents from tracenatural saponin impurities from the tree bark. As a result, it wasnecessary to explore whether there were any naturally derived tracesaponin impurities that may function as the immuno-active constituent.

Groups of five mice were immunized with the melanoma antigen GD3ganglioside conjugated to KLH (GD3-KLH) at a 10 μg antigen dose pervaccination. As the negative control, mice were vaccinated with theGD3-KLH antigen only. As a positive control, vaccinations were performedwith naturally derived NQS-21 (derived by fractionating a mixture ofsaponins from Quillaja saponaria) at a dose of 20 μg, an amount known toinduce measurable antibody responses with acceptable toxic effects inmice. Evaluation of the adjuvant activity of synthetic SQS-21 adjuvantsat the same dose (20 μg) included a reconstituted mixture of SQS-21-Apiand SQS-21-Xyl in a 65:35 mixture (SQS-21-Mix) to mimic typical isomericratios found within NQS-21. Similar evaluations were performed on eachof the separate saponin isomers, SQS-21-Api and SQS-21-Xyl at 20 μgdoses. Mice were immunized weekly for three weeks with the stated dosesof specified adjuvants, and were bled 10 days after the thirdvaccination. Immunopotentiation was assessed by testing for the presenceof antibody against GD3 and KLH by ELISA, and against a tumor cell-lineSK-MEL-28, expressing GD3 antigen, by FACS.

Antibody Response (ELISA).

The antibody response after vaccination with GD3-KLH conjugate with orwithout adjuvant was determined with an ELISA assay using either GD3ganglioside or KLH protein as target. Comparison of the differentadjuvants at the same doses (10 and 20 μg) of SQS-21, SQS-21-Api andSQS-21-Xyl were all equally effective at inducing and IgM antibodyresponse against GD3 after 3 weekly vaccinations with antibody titers ineach case significantly higher than the group with GD3-KLH alone. Afterthe third vaccination, no IgG antibodies against GD3 were detected;however, after the fourth immunization IgM and IgG antibody titers wereinduced in most mice. Again, there were no significant differencesbetween the various groups. The IgG antibody response against KLH wasalso strikingly elevated in all groups with no group demonstratingsignificantly higher or lower titers than the others. All were at least20 fold higher than the GD3-KLH alone group. A graphical representation(FIG. 2 ) of the antibody titers at 20 μg QS-adjuvant dose clearlyillustrates that the SQS-21 adjuvants possess comparable adjuvantactivity to that of NQS-21. FIGS. 2 a-c show Anti-GD3 and Anti-KLHantibody titers after vaccination with GD3-KLH conjugate (10 μg) withadjuvants NQS-21, SQS-21-Mix, SQS-21-Api, or SQS-21-Xyl each at 20 μgdoses. Each value represents median value of five mice (sera tested 7days after 3^(rd) and 4^(th) vaccination). NQS-21=naturally derivedQS-21; SQS-21-Mix=synthetic QS-21.

Cell Surface Reactivity by Flow Cytometry (FACS).

Immunopotentiation was also assessed by testing for the presence ofantibody against a tumor cell-line SK-MEL-28) expressing GD3 antigen byFACS. Sera drawn 7 days after the 4^(th) vaccination was tested for cellsurface reactivity by flow cytometry using the SK-Mel-28 (GD3 positive)cell line. The median FACS results are represented graphically at 20 μgQS-adjuvant dose for direct comparison (FIG. 3 ). Presera obtained frommice before immunization showed less than 10% positive cells and serafrom mice vaccinated with all three synthetic adjuvants showedsignificant positive reactivity with SK-Mel-28. These data reinforce thecomparable adjuvant activity of synthetic SQS-21 relative to that ofNQS-21.

Toxicity.

As a measure of toxicity, loss of weight was monitored at 0 h, 24 h, 48h and 72 h after each injection. The median weight loss for groups offive mice receiving SQS-21-Mix, SQS-21-Api or SQS-21-Xyl at the threedifferent doses is demonstrated in FIG. 4 . As the negative control,vaccinations employing GD3-KLH only (no adjuvant) led to no appreciableweight loss after each injection. As the positive control, the presenceof NQS-21 (20 μg) elicited notable and expected weight loss after eachinjection. This pattern of weight loss over the 4-week duration is verysimilar to that of the other groups of mice vaccinated with SQS-21synthetic adjuvants (mix, and separate isomers), again signaling acomparable biological profile of SQS-21 compared to that of NQS-21.

Example 5

Preclinical Evaluation of Selected Compounds of Formula II with GD3-KLHConjugate Vaccine.

Synthetic efforts have resulted in the preparation of compounds offormula H, including compounds I-8, I-9, and I-10. In these compounds,structural variations within the hydrolytically labile acyl chain ofnatural QS compounds take the form of significantly more stableamide-linked constructs, along with dramatically simplified linear alkylchain variants.

Groups of five mice were vaccinated (3×@1-wk intervals, booster @wk 7)with GD3-KLH antigen (10 μg) along with the adjuvant of interest (10μg). As a negative control, one group was vaccinated with the antigenonly, devoid of adjuvant. As positive controls, two groups werevaccinated with either natural NQS-21 or synthetic SQS-21-Mix, both ofwhich were established to be potent immunoadjuvants in this assay (seeFIG. 2 ). Three compounds of formula II, as well as our recentlysynthesized QS-7-Api adjuvant, were evaluated in parallel for comparison(FIG. 5 ).

Antibodies to GD3 in the blood samples were measured by ELISA (FIG. 5A).These preliminary data reveal that these synthetic compounds are atleast as active as NQS-21 in terms of immunopotentiating ability. Theseactivities are reinforced by the strikingly high Ab titers (0.8-1.6×10⁶,FIG. 5B) against the protein antigen KLH. Detailed toxicity assessmentof these novel adjuvants is currently underway.

This important collection of preliminary data (FIG. 5 ) establishes theconcepts that: (1) hydrolytically stable compounds of formula H areactive immunopotentiators; and (2) that the present invention providesnovel and potent adjuvant compounds with potencies that rival and exceedthat of natural NQS-21.

1-41. (canceled)
 42. A Compound selected from the group consisting of